Novel pgc-1 isoforms and uses therefor

ABSTRACT

The invention provides isolated nucleic acid molecules, designated PGC-1 b  and PGC-1 c  nucleic acid molecules, which encode novel isoforms of PGC-1 family members. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing PGC-1 nucleic acid molecules, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which a PGC-1 gene has been introduced or disrupted. The invention still further provides isolated PGC-1 proteins, fusion proteins, antigenic peptides and anti-PGC-1 antibodies. Diagnostic methods utilizing compositions of the invention are also provided.

RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication Ser. No. 60/303,468, filed Jul. 5, 2001, the entire contentsof which are incorporated herein by this reference.

GOVERNMENT SUPPORT

[0002] Work described herein was supported under grant DK54477 awardedby the National Institutes of Health. The U.S. government may havecertain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] Heart and skeletal muscle rely primarily on fatty acid oxidationto support their energy needs. Fatty acids are transported betweenorgans either as non-esterified fatty acid (NEFAs) or astriacylglycerols complexed to lipoproteins. Whereas the cellular uptakeof NEFAs is thought to be facilitated by a protein facilitated mechanisminvolving a broad class of transporters (e.g., FABPm, FAT/CD36, andFATP), the uptake of lipoprotein borne fatty acids first requires thehydrolysis of the ester bond through the action of lipoprotein lipase(LPL). After becoming activated in the cytosol by acyl CoA synthetase,fatty-acyl-CoAs enter the beta oxidation pathway. Although mitochondriaare the main site of oxidation for short and medium chain length fattyacids, longer chain fatty acids (C₂₂ and longer) are first oxidized inperoxisomes, until palmitoyl-CoA is formed, and further oxidized in themitochondria. In addition to this chain shortening activity, peroxisomalbeta oxidation is also responsible for the catabolism of a wide varietyof fatty acid analogues such as dicarboxylic acids and prostaglandins.

[0004] Several animal models have stressed the importance of fatty acidoxidation in different tissues. For example, mice deficient inperoxisomal Acyl-CoA oxidase accumulate very long chain fatty acids(VLCFAs) in the blood, exhibit severe steatohepatitis (inflammation ofthe liver associated with fatty liver), and ultimately develop livertumors (Fan, C. Y. et al. (1998) J. Biol. Chem. 273(25):15639-45). Thefatty liver phenotype is also observed in response to fasting in micelacking the transcription factor PPARα, highlighting its critical rolefor the induction of fatty acid oxidation enzymes in liver in responseto nutritional challenge (Hashimoto, T. et al. (2000) J. Biol. Chem.275(37):28918-28).

[0005] Conversely, uncontrolled fatty acid oxidation can also bedetrimental. Transgenic mice overexpressing LPL in skeletal muscle andheart have increased free fatty acid (FFA) uptake in muscle and lowerplasma triglyceride levels, but suffer from extensive mitochondrial andperoxisomal proliferation, leading to severe myopathy and prematuredeath (Levak-Frank, S. et al. (1995) J. Clin. Invest. 96(2):976-86).

[0006] Although skeletal muscle is quantitatively the most importanttissue for removing lipids from circulation, little is known about thefactors that regulate fatty acid metabolism in this tissue. Whilecompelling evidence supports the role of PPARα in the regulation offatty acid oxidation in liver, recent data have shown that PPARα is notrequired for the constitutive expression of genes involved inperoxisomal beta oxidation in muscle (Djouadi, F. et al. (1998) J. Clin.Invest. 102(6):1083-91; Aoyama, T. et al. (1998) J. Biol. Chem.273(10):5678-84; Watanabe, K. et al. (2000) J. Biol. Chem.275(29):22293-9).

SUMMARY OF THE INVENTION

[0007] The present invention is based, at least in part, on thediscovery of novel isoforms of the thermogenic co-activator PGC-1. Theseisoforms, referred to herein as PGC-1b and PGC-1c, encode two shorterproteins corresponding to the amino-terminal region of PGC-1, but lackthe entirety of its carboxy-terminal RNA processing domain.

[0008] In one embodiment, the invention features an isolated nucleicacid molecule that includes the nucleotide sequence set forth in SEQ IDNO:6, 8, 9, 11, 12, 14, 15, or 17, or a complement thereof. In anotherembodiment, the invention features an isolated nucleic acid moleculewhich encodes a polypeptide which includes the amino acid sequence setforth in SEQ ID NO:7, 10, 13, or 16.

[0009] In other embodiments, the invention features nucleic acidmolecules that are complementary to, antisense to, or hybridize understringent conditions to the isolated nucleic acid molecules describedherein.

[0010] In a related aspect, the invention provides vectors including theisolated nucleic acid molecules described herein (e.g., PGC-1-encodingnucleic acid molecules). Such vectors can optionally include nucleotidesequences encoding heterologous polypeptides. Also featured are hostcells including such vectors (e.g., host cells including vectorssuitable for producing PGC-1 nucleic acid molecules and polypeptides).

[0011] In another aspect, the invention features isolated PGC-1polypeptides and/or biologically active or antigenic fragments thereof.Exemplary embodiments feature an isolated polypeptide including theamino acid sequence of SEQ ID NO:7, 10, 13, or 16.

[0012] The PGC-1 polypeptides and/or biologically active or antigenicfragments thereof, are useful, for example, as reagents or targets inassays applicable to treatment and/or diagnosis of PGC-1 associateddisorders. In one embodiment, a PGC-1 polypeptide or fragment thereofhas a PGC-1 activity. In another embodiment, a PGC-1 polypeptide orfragment thereof has at least one or more of the following domains ormotifs: a tyrosine phosphorylation motif, a cAMP phosphorylation motif,an LXXLL motif, and/or a peroxisomal localization signal, andoptionally, has a PGC-1 activity. In a related aspect, the inventionfeatures antibodies (e.g., antibodies which specifically bind to any oneof the polypeptides, as described herein) as well as fusion polypeptidesincluding all or a fragment of a polypeptide described herein.

[0013] The present invention further features methods for detectingPGC-1 polypeptides and/or PGC-1 nucleic acid molecules, such methodsfeaturing, for example, a probe, primer or antibody described herein.Also featured are kits for the detection of PGC-1 polypeptides and/orPGC-1 nucleic acid molecules. In a related aspect, the inventionfeatures methods for identifying compounds which bind to and/or modulatethe activity of a PGC-1 polypeptide or PGC-1 nucleic acid moleculedescribed herein. Also featured are methods for modulating PGC-1activity.

[0014] Other features and advantages of the invention will be apparentfrom the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 depicts the nucleotide sequence of PGC-1b (SEQ ID NO:6).The unique region is underlined.

[0016]FIG. 2 depicts the amino acid sequence of PGC-1b (SEQ ID NO:7).The unique region is underlined.

[0017]FIG. 3 depicts the nucleotide sequence of PGC-1c (SEQ ID NO:12).The unique region is underlined.

[0018]FIG. 4 depicts the amino acid sequence of PGC-1c (SEQ ID NO:13).The unique region is underlined.

[0019]FIG. 5 depicts the structural relationship between the PGC-1a,PGC-1b, and PGC-1c nucleotide and amino acid sequences. The commonnucleotide region, most of which is shown at the top, corresponds tonucleotides 62-964 of SEQ ID NO:1 (PGC-1a), nucleotides 60-962 of SEQ IDNO:6 (PGC-1b), and 37-939 of SEQ ID NO:12 (PGC-1c). Only the last 30nucleotides of the 5′ untranslated region (immediately prior to theinitiation codon) are shown. The common amino acid sequence (set forthas SEQ ID NO:5) corresponds to amino acid residues 1-291 of SEQ ID NO:2(PGC-1a), SEQ ID NO:7 (PGC-1c), and SEQ ID NO:13 (PGC-1c). The PGC-1aspecific sequences correspond to nucleotides 965-3066 of SEQ ID NO:1 andamino acid residues 292-797 of SEQ ID NO:2 (however, the entire specificnucleotide and amino acid sequences of PGC-1a are not shown in thefigure). The PGC-1b specific sequences correspond to nucleotides963-1893 of SEQ ID NO:6 (set forth as SEQ ID NO:9) and amino acidresidues 292-320 of SEQ ID NO:7 (set forth as SEQ ID NO:10). The PGC-1cspecific sequences correspond to nucleotides 940-1744 of SEQ ID NO:12(set forth as SEQ ID NO:15) and amino acid residues 292-300 of SEQ IDNO:13 (set forth as SEQ ID NO:16).

[0020]FIG. 6 depicts an alignment of the 5′ UTRs of PGC-1a, PGC-1b, andPGC-1c, corresponding to nucleotides 1-91 of SEQ ID NO:1 (PGC-1a),nucleotides 1-89 of SEQ ID NO:6 (PGC-1b), and nucleotides 1-66 of SEQ IDNO:12 (PGC-1c).

[0021]FIG. 7 depicts a schematic representation of the structuralrelationship between the nucleotide sequences of PGC-1a, PGC-1b, andPGC-1c. The coding regions are boxed. Open boxes represent the codingregion common to all three isoforms, while hatched boxes represent thecoding regions specific to each isoform. Dashed lines indicate points ofdivergence between the sequences of each isoform.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention is based, at least in part, on thediscovery of novel isoforms of the thermogenic co-activator PGC-1. Theseisoforms, referred to herein as PGC-1b and PGC-1c, encode two shorterproteins corresponding to the amino-terminal region of PGC-1, but lackthe entirety of its carboxy-terminal RNA processing domain.

[0023] PGC-1 has been described previously (Puigserver, P. et al. (1998)Cell 92(6):829-39; U.S. Pat. No. 6,166,192; and PCT InternationalPublication Nos. WO 98/54220; the contents of all of which areincorporated herein by reference). Based on the instant discovery ofnovel isoforms of PGC-1, the molecule previously referred to as “PGC-1”is referred to herein as “PGC-1a”, and the term “PGC-1”, as used herein,includes PGC-1a, PGC-1b, and PGC-1c. The nucleotide and amino acidsequences of PGC-1b are shown in FIGS. 1 and 2, respectively. Thenucleotide and amino acid sequences of PGC-1c are shown in FIGS. 3 and4, respectively. FIGS. 5 and 7 depict the structural relationshipbetween PGC-1a, PGC-1b, and PGC-1c.

[0024] PGC-1b and PGC-1c are identical to PGC-1a at the 5′ end of thenucleic acid sequence, although there are slight differences at theextreme 5′ ends of the 5′ untranslated regions (UTRs) of each isoform(see FIG. 6). Accordingly, the region common to all three isoforms(referred to herein as the “common region”; set forth as SEQ ID NO:4)corresponds to nucleotides 41-964 of PGC-1a (SEQ ID NO:1), nucleotides39-962 of PGC-1b (SEQ ID NO:6), and nucleotides 16-939 of PGC-1c (SEQ IDNO:12). The nucleotide sequences of PGC-1b and PGC-1c each containunique regions located 3′ to the common region. The amino acid sequencecommon to all three isoforms (set forth as SEQ ID NO:5) corresponds toresidues 1-291 of PGC-1a (SEQ ID NO:2), PGC-1b (SEQ ID NO:7), and PGC-1c(SEQ ID NO:13), and the unique region of each polypeptide starts atamino acid residue 292.

[0025] The unique region of PGC-1b corresponds to nucleotides 963-1893of SEQ ID NO:6 (set forth as. SEQ ID NO.9). The entire coding region ofPGC-1b, corresponding to nucleotides 90-1049 of SEQ ID NO:6, is setforth as SEQ ID NO:8 and encodes a polypeptide of 320 amino acidresidues (set forth as SEQ ID NO:7). The unique coding region of PGC-1b,corresponding to nucleotides 963-1049 of SEQ ID NO:6, is set forth asSEQ ID NO:11 and encodes a unique C-terminus of 29 amino acid residues(set forth as SEQ ID NO:10).

[0026] The unique region of PGC-1c corresponds to nucleotides 940-1744of SEQ ID NO:12 (set forth as SEQ ID NO:15). The entire coding region ofPGC-1c, corresponding to nucleotides 67-966 of SEQ ID NO:12, is setforth as SEQ ID NO:14 and encodes a polypeptide of 300 amino acidresidues (set forth as SEQ ID NO:13). The unique coding region ofPGC-1c, corresponding to nucleotides 940-966 of SEQ ID NO:12, is setforth as SEQ ID NO:17 and encodes a unique C-terminus of 9 amino acidresidues (set forth as SEQ ID NO:16). It should be noted thatnucleotides 940-1648 of PGC-1c (SEQ ID NO:12) are actually present inthe nucleotide sequence of PGC-1b (from nucleotides 1092-1859 of SEQ IDNO:6), but are not translated therein.

[0027] The instant discoveries indicate that unlike expression ofPGC-1a, the expression of the PGC-1b isoform in C2C12 myotubes does notaffect mitochondrial function, either directly or through aninterference with PGC-1a However, PGC-1b induced the expression ofseveral genes that regulate fatty acid uptake and utilization. The genesactivated by PGC-1b in C2C12 cells are similar to the genes that areinduced in skeletal muscle upon fasting, a condition known to shiftmuscle substrate utilization from glucose to fatty acids. Surprisingly,while some of these genes were induced by both PGC-1a and PGC-1b,lipoprotein lipase (LPL) and Acyl Co-A oxidase (AOX) were only inducedby PGC-1b.

[0028] The-transcriptional co-activator PGC-1a plays a major role inregulating critical aspects of cellular metabolism. When expressed inmuscle cells, PGC-1a stimulates mitochondrial biogenesis andsimultaneously activates UCP-2, a mitochondrial membrane proteinbelonging to the uncoupling protein family (Wu, Z. et al. (I999) Cell98(1): 115-24). In addition to stimulating the mitochondrial capacity inmuscle cells, PGC-1a concurrently stimulates the expression of Glut4,resulting in increased glucose uptake (Michael, L. F. et al. (2001)Proc. Natl. Acad. Sci. USA 98(7):3820-5). Furthermore, PGC-1a was alsoshown to stimulate the expression in cardiomyocytes of MCAD and M-CPT-1,two regulatory factors of the mitochondrial beta oxidation pathway(Lehman, J. J. et al. (2000) J. Clin. Invest. 106(7):847-56).

[0029] The novel isoforms, PGC-1b and PGC-1c, provide novel targets forthe regulation of cellular metabolism, including fatty acid metabolism.While both isoforms are excluded from the nucleus, PGC-1b, owing to itscarboxy-terminal peroxisomal localization signal, is targeted toperoxisomes. In contrast to PGC-1a, PGC-1b lacks the ability tostimulate mitochondrial biogenesis and function in C2C12 myoblasts.However, this isoform specifically activates several critical genesinvolved in the uptake and utilization of fatty acids.

[0030] The rate of fatty acid oxidation in muscle is an important factoraffecting whole-body metabolism. Altering this variable can haveprofound influence on pathologies such as obesity, diabetes and heartdisease. This rate has been shown to be directly stimulated by severalhormones, including thyroid and growth hormones, and more recently byadipsin, a protein secreted by the adipose tissue (Fruebis, J. et al.(2001) Proc. Natl. Acad. Sci. USA 98(4):2005-2010). In addition to thesehormonal cues, the rate of fatty acid oxidation has also been shown tobe regulated by the metabolic intermediate malonyl-CoA (Abu-Elheiga, L.et al. (2001) Science 291(5513):2613-6). Mice that were engineered toproduce less malonyl-CoA in muscle (through disruption of the enzymeAcyl-CoA carboxylase (ACC)) had lower adiposity and exhibited anincreased ability to oxidize fatty-acid in skeletal muscle as expected,but were also found to be hyperphagic, a phenotype that is strikinglysimilar to mice overexpressing the uncoupling protein UCP3 in muscle(Clapham, J. C. et al. (2000) Nature 406(6794):415-8). PGC-1b activatesmalonyl CoA dehydrogenase (MCD), an enzyme whose action counters theeffect of ACC, thereby promoting the flux of fatty acid through the betaoxidation pathway in mitochondria.

[0031] While increasing skeletal muscle fatty acid uptake byoverexpressing LPL had a beneficial effect on adiposity in mice, itproved to be detrimental to muscle physiology if not accompanied by aparallel increase in fatty acid oxidation. Because PGC-1b appears tostimulate the whole program of fatty acid uptake and utilization, thismolecule may represent a target for the development of anti-obesitydrugs.

[0032] The term “family” when referring to the protein and nucleic acidmolecules of the invention is intended to mean two or more proteins ornucleic acid molecules having a common structural domain or motif andhaving sufficient amino acid or nucleotide sequence homology as definedherein. Such family members can be naturally or non-naturally occurringand can be from either the same or different species. For example, afamily can contain a first protein of human origin as well as otherdistinct proteins of human origin or alternatively, can containhomologues of non-human origin, e.g., rat or mouse proteins. Members ofa family can also have common functional characteristics.

[0033] For example, the family of PGC-1 proteins, including the PGC-1proteins of the present invention, include several domains and/ormotifs. These domains/motifs include: a putative tyrosinephosphorylation site (at amino acid residues 204-212 of SEQ ID NO:7 andamino acid residues 204-212 of SEQ ID NO: 13), a putative cAMPphosphorylation site (at amino acid residues 238-241 of SEQ ID NO:7 andamino acid residues 238-241 of SEQ ID NO:13), an LXXLL motif (at aminoacids 142-146 of SEQ ID NO:7 and amino acid residues 142-146 of SEQ IDNO:13) which mediates interaction with a nuclear receptor, and aperoxisomal localization signal (at amino acid residues 318-320 of SEQID NO:7). As used herein, a tyrosine phosphorylation site is an aminoacid sequence which includes at least one tyrosine residue which can bephosphorylated by a tyrosine protein kinase. Typically, a tyrosinephosphorylation site is characterized by a lysine or an arginine aboutseven residues to the N-terminal side of the phosphorylated tyrosine. Anacidic residue (asparagine or glutamine) is often found at either threeor four residues to the N-terminal side of the tyrosine (Patschinsky, T.et al. (1982) Proc. Natl. Acad. Sci. USA 79:973-977); Hunter, T. (1982)J. Biol. Chem. 257:4843 -4848; Cooper, J. A. et al. (1984) J. Biol.Chem. 259:7835-7841). As used herein, a cAMP phosphorylation site is anamino acid sequence which includes a serine or threonine residue whichcan be phosphorylated by a cAMP-dependent protein kinase. Typically, thecAMP phosphorylation site is characterized by at least two consecutivebasic residues to the N-terminal side of the serine or threonine(Fremisco, J. R. et al. (1980) J. Biol. Chem. 255:42404245; Glass, D. B.and Smith, S. B. (1983) J. Biol. Chem. 258:14797-14803; Glass, D. B. etal. (1986) J. Biol. Chem. 261:2987-2993). As used herein, an LXXLL motifrefers to a motif wherein X can be any amino acid and which mediates aninteraction between a nuclear receptor and a coactivator (Heery et al.(1997) Nature 397:7-33-736; Torchia et al. (1997) Nature 387:677-684).As used herein, a peroxisomal localization signal refers to a proteinmotif that, when present in a protein, is sufficient to directlocalization of said protein to peroxisomes.

[0034] Isolated proteins of the present invention, preferably PGC-1proteins, have an amino acid sequence sufficiently homologous to theamino acid sequence of SEQ ID NO:7, 10, 13, or 16, or are encoded by anucleotide sequence sufficiently homologous to SEQ ID NO:6, 8, 9, 11,12, 14, 15, or 17. As used herein, the term “sufficiently homologous”refers to a first amino acid or nucleotide sequence which contains asufficient or minimum number of identical or equivalent (e.g., an aminoacid residue which has a similar side chain) amino acid residues ornucleotides to a second amino acid or nucleotide sequence such that thefirst and second amino acid or nucleotide sequences share commonstructural domains or motifs and/or a common functional activity. Forexample, amino acid or nucleotide sequences which share commonstructural domains having at least 60%, 65%, 70%, 75%, 80%, 85%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.25%, 99.5%, 99.75%or more homology or identity across the amino acid sequences of thedomains and contain at least one and preferably two structural domainsor motifs, are defined herein as sufficiently homologous. Furthermore,amino acid or nucleotide sequences which share at least 60%, 65%, 70%,75%, 80%, 85%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.25%, 99.5%, 99.75% or more homology or identity and share a commonfunctional activity are defined herein as sufficiently homologous.

[0035] In a preferred embodiment, a PGC-1 protein includes at least oneor more of the following domains or motifs: a tyrosine phosphorylationmotif, a cAMP phosphorylation motif, an LXXLL motif, and/or aperoxisomal localization signal, and has an amino, acid sequenceat leastabout 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.25%, 99.5%, 99.75% or more homologous oridentical to the amino acidsequence of SEQ ID NO:7, 10, 13, or 16, orthe amino acid sequence encoded by the DNA insert of the plasmiddeposited with ATCC as Accession Number ______ or ______. In yet anotherpreferred embodiment, a PGC-1 protein includes at least one or more ofthe following domains or motifs: a tyrosine phosphorylation motif, acAMP phosphorylation motif, an LXXLL motif, and/or a peroxisoqmallocaliation signal, and is encoded by a nucleic acid molecule having anucleotide sequence which hybridizes under stringent hybridizationconditions to a complement of a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:6, 8, 9, 11, 12, 14, 15, or 17. Inanother preferred embodiment, a PGC-1 protein includes at least one ormore of the following domains or motifs: a tyrosine phosphorylationmotif, a cAMP phosphorylation motif, an LXXLL motif, and/or aperoxisomal localization signal, and has a PGC-1 activity.

[0036] As used interchangeably herein, a “PGC-1 activity”, “biologicalactivity of PGC-1” or “functional activity of PGC-1”, includes anactivity exerted or mediated by a PGC-1 protein, polypeptide or nucleicacid molecule when expressed in a cell or on a membrane, as determinedin vivo or in vitro, according to standard techniques. In oneembodiment, a PGC-1 activity is a direct activity, such as interactionwith a PGC-1 target molecule. In another embodiment, a PGC-1 activity isan indirect activity mediated, for example, by interaction of a PGC-1molecule with a PGC-1 target molecule or binding partner. As usedherein, a “target molecule” or “binding partner” is a molecule withwhich a PGC-1 protein binds or interacts in nature, such that functionof the target molecule or binding partner is modulated. In an exemplaryembodiment, a PGC-1 target molecule or binding partner is aperoxisomalprotein.

[0037] In a preferred embodiment, a PGC-1 activity is at leastone of thefollowing activities: (i) interaction with a PGC-1 target molecule; (ii)modulation of intracellular signaling; (iii) modulation of cellularmetabolism; (iv) localization to peroxisomes; (v) modulation of theexpression of genes involved in fatty acid uptake and/or oxidation(e.g., LPL, FAT/CD36, VLACS, AOX, MCAD, and/or MCD); (vi) modulation offatty acid uptake and/or oxidation; (vii) modulation of energyhomeostasis; and/or (viii) modulation of lipid homeostasis.

[0038] The nucleotide sequence of the isolated human PGC-1b cDNA and thepredicted amino acid sequence encoded by the PGC-1b cDNA are shown inFIGS. 1 and 2, respectively, and in SEQ ID NOs:6 and 7, respectively. Aplasmid containing the huan PGC-1 cDNA was deposited with the AmericanType Culture Collection (ATCC), 10801 University. Boulevard, Manassas,Va. 20110-2209, on ______ and assigned Accession Number ______. Thisdeposit will be maintained under the terms of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure. This deposit were made merely as aconvenience for those of skill in the art andis not an admission that adeposit is required under 35 U.S.C. §112.

[0039] The human PGC-1b gene, which is approximately 1893 nucleotides inlength, encodes a protein having a molecular weight of approximately35.2 kD which is approximately 320 amino acid residues in length.

[0040] The nucleotide sequence of the isolated human PGC-1c cDNA and thepredicted amino acid sequence encoded by the PGC-1 cDNA are shown inFIGS. 3 and 4, respectively, and in SEQ ID NOs:12 and 13, respectively.A plasmid containing the human PGC-1c cDNA was deposited with theAmerican Type Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va. 20110-2209, on ______ and assigned Accession Number______. This deposit will be maintained under the terms of the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purposes of Patent Procedure. This deposit were made merely as aconvenience for those of skill in the art and is not an admission that adeposit is required under 35 U.S.C. §112.

[0041] The human PGC-1c gene, which is approximately 1744 nucleotides inlength, encodes a protein having a molecular weight of approximately 33kD which is approximately 300 amino acid residues in length.

[0042] Various aspects of the invention are described in further detailin the following subsections:

[0043] I. Isolated Nucleic Acid Molecules

[0044] One aspect of the invention pertains to isolated nucleic acidmolecules that encode PGC-1 proteins or biologically active portionsthereof, as well as nucleic acid fragments sufficient for use ashybridization probes to identity PGC-1-encoding nucleic acidmolecules.(e.g., PGC-1 mRNA) and fragments for use as PCR primers forthe amplification or mutation of PGC-1 nucleic acid molecules. As usedherein, the term “nucleic acid molecule” is intended to include DNAmolecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) andanalogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA.

[0045] The term “isolated nucleic acid molecule” includes nucleic acidmolecules which are separated from other nucleic acid molecules whichare present in the natural source of the nucleic acid. For example, withregards to genomic DNA, the term “isolated” includes nucleic acidmolecules which are separated from the chromosome with which the genomicDNA is naturally associated. Preferably, an “isolated” nucleic acid isfree of sequences which naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated PGC-1 nucleic acidmolecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5kb or 0.1 kb of nucleotide sequences which naturally flank the nucleicacid molecule in genomic DNA of the cell from which the nucleic acid isderived. Moreover, an “isolated” nucleic acid molecule, such as a cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized.

[0046] A nucleic acid molecule of the present invention, e.g., a nucleicacid molecule having the nucleotide sequence of SEQ ID NO:6, 8, 9, 11,12, 14, 15, or 17, or the nucleotide sequence of the DNA insert of theplasmid deposited with ATCC as Accession Number ______ or ______, or aportion thereof, can be isolated using standard molecular biologytechniques and the sequence information provided herein. Using all or aportion of the nucleic acid sequence of SEQ ID NO:6, 8, 9, 11, 12, 14,15, or 17, or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number ______ or ______, ashybridization probes; PGC-1 nucleic acid molecules can be isolated usingstandard hybridization and cloning techniques (e.g., as described inSambrook, J. et al., Molecular Cloning: A Laboratory Manual. 2^(nd) ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989).

[0047] Moreover, a nucleic acid molecule encompassing all or a portionof SEQ ID NO:6, 8, 9, 11, 12, 14, 15, or 17, or the nucleotide sequenceof the DNA insert of the plasmid deposited with ATCC as Accession Number______ or ______ can be isolated by the polymerase chain reaction (PCR)using synthetic oligonucleotide primers designed based upon the sequenceof SEQ ID NO:6, 8, 9, 11, 12, 14, 15, or 17, or the nucleotide sequenceof the DNA insert of the plasmid deposited with ATCC as Accession Number______ or ______.

[0048] A nucleic acid of the invention can be amplified using cDNA, mRNAor alternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to PGC-1 nucleotidesequences can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

[0049] In one embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO:6, 8, 9,11, 12, 14, 15, or 17. This cDNA may comprise sequences encoding thehuman PGC-1b protein (e.g., the “coding region”, from nucleotides90-1049), as well as 5′ untranslated sequence (nucleotides 1-89) and 3′untranslated sequences (nucleotides 1050-1893) of SEQ ID NO:6.Alternatively, the nucleic acid molecule can comprise only the codingregion of SEQ ID NO:6 (e.g., nucleotides 90-1049, corresponding to SEQID NO:8). Accordingly, in another embodiment, an isolated nucleic acidmolecule of the invention comprises SEQ ID NO:8 and nucleotides 1-89 ofSEQ ID NO:6. In yet another embodiment, the isolated nucleic acidmolecule comprises SEQ ID NO:8 and nucleotides 1050-1893 of SEQ ID NO:6.In yet another embodiment, the nucleic acid molecule consists of thenucleotide sequence set forth as SEQ ID NO:6 or SEQ ID NO:8. In anotherembodiment, an isolated nucleic acid molecule of the invention comprisesSEQ ID NO:8 and a stop codon (e.g., nucleotides 1050-1052 of SEQ IDNO:6).

[0050] In another embodiment, an isolated nucleic acid molecule of theinvention comprises the “unique region” of the human PGC-1b gene (e.g.,the “unique coding region”, from nucleotides 963-1049, as well as 3′untranslated sequences (nucleotides 1050-1893) of SEQ ID NO:6),corresponding to SEQ ID NO:9. Alternatively, the nucleic acid moleculecan comprise only the unique coding region of SEQ ID NO:6 (e.g.,nucleotides 963-1049, corresponding to SEQ ID NO: 11). In anotherembodiment, the nucleic acid molecule consists of the nucleotidesequence set forth as SEQ ID NO:9 or SEQ ID NO:11. In anotherembodiment, an isolated nucleic acid molecule of the invention comprisesSEQ ID NO:11 and a stop codon (e.g., nucleotides 1050-1052 of SEQ IDNO:6). In other embodiments, an isolated nucleic acid molecule of theinvention comprises nucleotides 1-38 or nucleotides 1-89 of SEQ ID NO:6.In still other embodiments, an isolated nucleic acid molecule of theinvention comprises nucleotides 1050-1893, nucleotides 963-1091,nucleotides 1092-1859, or nucleotides 1860-1893 of SEQ ID NO:6.

[0051] In another embodiment, the cDNA may comprise sequences encodingthe human PGC-1c protein (e.g., the “coding region”, from nucleotides67-966), as well as 5′ untranslated sequence (nucleotides 1-66) and 3′untranslated sequences (nucleotides 967-1744) of SEQ ID NO: 12.Alternatively, the nucleic acid molecule can comprise only the codingregion of SEQ ID NO:12 (e.g., nucleotides 67-966, corresponding to SEQID NO:14). Accordingly, in another embodiment, an isolated nucleic acidmolecule of the invention comprises SEQ ID NO:14 and nucleotides 1-66 ofSEQ ID NO:12. In yet another embodiment, the isolated nucleic acidmolecule comprises SEQ ID NO:14 and nucleotides 967-1744 of SEQ IDNO:12. In yet another embodiment, the nucleic acid molecule consists ofthe nucleotide sequence set forth as SEQ ID NO: 12 or SEQ ID NO:14. Inanother embodiment, an isolated nucleic acid molecule of the inventioncomprises SEQ ID NO:14 and a stop codon (e.g., nucleotides 967-969 ofSEQ ID NO:12).

[0052] In another embodiment, an isolated nucleic acid molecule of theinvention comprises the “unique region” of the human PGC-1c gene (e.g.,the “unique coding region”, from nucleotides 940-966, as well as 3′untranslated sequences (nucleotides 967-1744) of SEQ ID NO:12),corresponding to SEQ ID NO:15. Alternatively, the nucleic acid moleculecan comprise only the unique coding region of SEQ ID NO:12 (e.g.,nucleotides 940-966, corresponding to SEQ ID NO:17). In anotherembodiment, the nucleic acid molecule consists of the nucleotidesequence set forth as SEQ ID NO:15 or SEQ ID NO:17. In otherembodiments, an isolated nucleic acid molecule of the inventioncomprises nucleotides 1-15 or nucleotides 1-66 of SEQ ID NO:12. In stillother embodiments, an isolated nucleic acid molecule of the inventioncomprises nucleotides 940-1068, nucleotides 967-1744, nucleotides967-1068, or nucleotides 1069-1744 of SEQ ID NO: 12.

[0053] In still another embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which is a complement ofthe nucleotide sequence shown in SEQ ID NO:6, 8, 9, 11, 12, 14,15, or17, or the nucleotide sequence of the

[0054] DNA insert of the plasmid deposited with ATCC as Accession Number______ or ______, or a portion of any of these nucleotide sequences. Anucleic acid molecule which is complementary to the nucleotide sequenceshown in SEQ ID NO:6, 8, 9, 11, 12, 14, 15, or 17, or the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC asAccession Number ______ or ______, is one which is sufficientlycomplementary to the nucleotide sequence shown in SEQ ID NO:6, 8, 9, 11,12, 14, 15, or 17, or the nucleotide sequence of the DNA insert of theplasmid deposited with ATCC as Accession Number ______ or ______, suchthat it can hybridize to the nucleotide sequence shown in SEQ ID NO:6,8, 9, 11, 12, 14, 15, or 17, or the nucleotide sequence of the DNAinsert of the plasmid deposited with ATCC as Accession Number ______ or______, thereby forming a stable duplex.

[0055] In still another embodiment,an isolated nucleic acid molecule ofthe present invention comprises a nucleotide sequence which is at leastabout 60%, 65%, 70%, 75%, 80%, 85%,.85%,.90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.25%, 99.5%, 99.75%, 99.5%, 99.75% or moreidentical to the nucleotide sequence shown in SEQ ID NO:6, 8, 9, 11, 12,14, 15, or 17 (e.g., to the entire length of the nucleotide sequence),or to the nucleotide sequence (e.g., the entire length of the nucleotidesequence) of the DNA insert of the plasmid deposited with ATCC asAccession Number ______ or ______, or a portion or complement of any ofthese nucleotide sequences. In one embodiment, anucleic acid molecule ofthe present invention comprises a nucleotide sequence which is at least(or no greater than) 25, 26, 27, 28, 30, 30, 36, 38, 40, 50, 59, 61, 87,100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 704,750, 800, 843, 850, 873, 874, 875, 876, 877, 900, 904, 905, 206, 906,907, 908, 909, 910, 911, 924, 925, 926, 927, 932, 933, 934, 939, 940,941, 947, 948, 949, 950, 1000, 1150, 1200, 1250, 1300, 1350, 1400, 1450,1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850 or more nucleotides inlength and hybridizes under stringent hybridization conditions to acomplement of a nucleic acid molecule of SEQ ID NO:6, 8, 9, 11, 12, 14,15, or 17, or the nucleotide. sequence ofthe DNA insert of the plasmiddeposited with ATCC as Accession Number ______ or ______. In anotherembodiment, a nucleic acid molecule of the present invention comprises afragment of at least 30-nucleotides, e.g., contiguous nucleotides, of anucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:6and includes at least nucleotide 964 of SEQ ID NO:6. In anotherembodiment, a nucleic acid molecule of the present invention comprises afragment of at least 30 nucleotides, e.g., 30 contiguous nucleotides, ofa nucleic acid molecule comprising the nucleotide sequence of SEQ IDNO:12 and includes at least nucleotide 942 of SEQ ID NO:12.

[0056] Moreover, the nucleic acid molecule of the invention can compriseonly a portion of the nucleic acid sequence of SEQ ID NO:6, 8, 9, 11,12, 14, 15, or 17, or the nucleotide sequence of the DNA insert of theplasmid deposited with ATCC as Accession Number _____ or ______, forexample, a fragment which can be used as a probe or primer or a fragmentencoding a portion of a PGC-1 protein, e.g., a biologically activeportion of a PGC-1 protein. The nucleotide sequence determined from thecloning of the PGC-1 gene allows for the generation of probes andprimers designed for use in identifying and/or cloning other PGC-1family members, as well as PGC-1 homologues from other species. Theprobe/primer (e.g., oligonucleotide) typically comprises substantiallypurified oligonucleotide. The oligonucleotide typically comprises aregion of nucleotide sequence that hybridizes under stringent conditionsto at least about 12or 15, preferably about 20 or 25, more preferablyabout 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of asense sequence of SEQ ID NO:6, 8, 9, 11, 12, 14, 15, or 17, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number ______ or ______, of an anti-sense sequence of SEQID NO:6, 8, 9, 11, 12, 14, 15, or 17, or the nucleotide sequence of theDNA insert of the plasmid deposited with ATCC as Accession Number ______or ______, or of a naturally occurring allelic variant or mutant of SEQID NO:6, 8, 9, 11, 12, 14, 15, or 17, or the nucleotide sequence of theDNA insert of the plasmid deposited with ATCC as Accession Number ______or ______.

[0057] Exemplary probes or primers are at least (or no greater than) 12or 15, 20 or 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or morenucleotides in length and/or comprise consecutive nucleotides of anisolated nucleic acid molecule described herein. Also included withinthe scope of the present invention are probes or primers comprisingcontiguous or consecutive nucleotides of an isolated nucleic acidmolecule described herein, but for the difference of 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 bases within the probe or primer sequence. Probes based onthe PGC-1 nucleotide sequences can be used to detect (e.g., specificallydetect) transcripts or genomic sequences encoding the same or homologousproteins. In preferred embodiments, the probe further comprises a labelgroup attached thereto, e.g. the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. In anotherembodiment a set of primers is provided, e.g., primers suitable for usein a PCR, which can be used to amplify a selected region of a PGC-1sequence, e.g., a domain, region, site or other sequence describedherein. The primers should be at least 5, 10, or 50 base pairs in lengthand less than 100, or less than 200, base pairs in length. The primersshould be identical, or differ by no greater than 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 bases when compared to a sequence disclosed herein or to thesequence of a naturally occurring variant. Such probes can be used as apart of a diagnostic test kit for identifying cells or tissue whichmisexpress a PGC-1 protein, such as by measuring a level of aPGC-1-encoding nucleic acid in a sample of cells from a subject, e.g.,detecting PGC-1 mRNA levels or determining whether a genomic PGC-1 genehas been mutated or deleted.

[0058] A nucleic acid fragment encoding a “biologically active portionof a PGC-1 protein” can be prepared by isolating a portion of thenucleotide sequence of SEQ ID NO:6, 8, 9, 11, 12, 14, 15, or 17, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number ______ or ______, which encodes a polypeptide havinga PGC-1 biological activity (the biological activities of the-PGC-1proteins are described herein), expressing the encoded portion of thePGC-1 protein (e g., by recombinant expression in vitro) and assessingthe activity of the encoded portion of the PGC-1 protein. In anexemplary embodiment, the nucleic acid molecule is at least 25, 26, 27,28, 30, 30, 36, 38, 40, 50, 59, 61, 87, 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 704, 750, 800, 843, 850, 873, 874,875, 876, 877, 900, 904, 905, 906, 906, 907, 908, 909, 910, 911, 924,925, 926, 927, 932, 933, 934, 939, 940, 941, 947, 948, 949, 950, 1000,1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700,1750, 1800, 1850 or more nucleotides in length and encodes a proteinhaving a PGC-1 activity (as described herein).

[0059] In another embodiment, the nucleic acid molecule encodes afragment of a polypeptide comprising the amino acid sequence of SEQ IDNO:10, wherein the fragment comprises at least 10-15, 15-20, 20-25,25-28 or more contiguous amino acid residues of the amino acid sequenceof SEQ ID NO:10. In another embodiment, the nucleic acid moleculeencodes a fragment of a polypeptide comprising the amino acid sequenceof SEQ ID NO:7 or 13, wherein the fragment comprises at least 9, 10, 15,20, 25, 29, 30, 31, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 225,250, 275, 291, 292, 293, 294, 295, 300 or more contiguous amino acidresidues of SEQ ID NO:7 or 13. In yet another embodiment, the nucleicacid molecule encodes a fragment of a polypeptide comprising the aminoacid sequence of SEQ ID NO:7, wherein the fragment comprises at least 9,10, 15, 20, 25, 29, 30, 31, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200,225, 250, 275, 292, 293, 294, 295, 300 or more contiguous amino acidresidues of SEQ ID NO:7 and includes at least residue 292 of SEQ IDNO:7. In still another embodiment, the nucleic acid molecule encodes afragment of a polypeptide comprising the amino acid sequence of SEQ IDNO:13, wherein the fragment comprises at least 9, 10, 15, 20, 25, 29,30, 31, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 292,293, 294, 295 or more contiguous amino acid residues of SEQ ID NO:13 andincludes at least residue 293 of SEQ ID NO:13.

[0060] The invention further encompasses nucleic acid molecules thatdiffer from the nucleotide sequence shown in SEQ ID NO:6, 8, 9, 11, 12,14, 15, or 17, or the nucleotide sequence of the DNA insert of theplasmid deposited with ATCC as Accession Number ______ or ______, due todegeneracy of the genetic code and thus encode the same PGC-1 proteinsas those encoded by the nucleotide sequence shown in SEQ ID NO:6, 8, 9,11, 12, 14, 15, or 17, or the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number _____ or ______. Inanother embodiment, an isolated nucleic acid molecule of the inventionhas a nucleotide sequence encoding a protein having an amino acidsequence which differs by at least 1, but no greater than 5, 10, 20, 50or 100 amino acid residues from the amino acid sequence shown in SEQ IDNO:7, 10, 13, or 16, or the amino acid sequence encoded by the DNAinsert of the plasmid deposited with the ATCC as Accession Number ______or ______. In yet another embodiment, the nucleic acid molecule encodesthe amino acid sequence of human PGC-1. If an alignment is needed forthis comparison, the sequences should be aligned for maximum homology.

[0061] Nucleic acid variants can be naturally occurring such as allelicvariants (same locus), homologues (different locus), and orthologues(different organism) or can be non naturally occurring. Non-naturallyoccurring variants can be made by mutagenesis techniques, includingthose applied to polynucleotides, cells, or organisms. The variants cancontain nucleotide substitutions, deletions, inversions and insertions.Variation can occur in either or both the coding and non-coding regions.The variations can produce both conservative and non-conservative aminoacid substitutions (as compared in the encoded product).

[0062] Allelic variants result, for example, from DNA sequencepolymorphisms within a population (e.g., the human population) that leadto changes in the amino acid sequences of the PGC-1 proteins. Suchgenetic polymorphism in the PGC-1 genes may exist among individualswithin a population due to natural allelic variation. As used herein,the terms “gene” and “recombinant gene” refer to nucleic acid moleculeswhich include an open reading frame encoding a PGC-1 protein, preferablya mammalian PGC-1 protein, and can further include non-coding regulatorysequences, and introns.

[0063] Accordingly, in one embodiment, the invention features isolatednucleic acid molecules which encode a naturally occurring allelicvariant of a polypeptide comprising the amino acid sequence of SEQ IDNO:7, 10, 13, or 16, or an amino acid sequence encoded by the DNA insertof the plasmid deposited with ATCC as Accession Number or wherein thenucleic acid molecule hybridizes to a complement of a nucleic acidmolecule comprising SEQ ID NO:6, 8, 9, 11, 12, 14, 15, or 17, forexample, under stringent hybridization conditions.

[0064] Allelic variants of PGC-1, e.g., human PGC-1, include bothfunctional and non-functional PGC-1 proteins. Functional allelicvariants are naturally occurring amino acid sequence variants of thePGC-1 protein that maintain the ability to, e.g., modulate expression ofgenes involved in fatty acid uptake and/or oxidation. Functional allelicvariants will typically contain only conservative substitution of one ormore amino acids of SEQ ID NO:7, 10, 13, or 16, or substitution,deletion or insertion of noncritical residues in non-critical regions ofthe protein.

[0065] Non-functional allelic variants are naturally occurring aminoacid sequence variants of the PGC-1 protein, e.g., human PGC-1, that donot have the ability to, e.g., modulate expression of genes involved infatty acid uptake and/or oxidation. Non-functional allelic variants willtypically contain a non-conservative substitution, a deletion, orinsertion, or premature truncation of the amino acid sequence of SEQ IDNO:7, 10, 13, or 16, or a substitution, insertion, or deletion incritical residues or critical regions of the protein.

[0066] The present invention further provides non-human orthologues(e.g., non-human orthologues of the human PGC-1 protein). Orthologues ofthe human PGC-1 protein are proteins that are isolated from non-humanorganisms and possess the same fatty acid uptake and/or oxidationmodulating activities as human PGC-1. Orthologues of the human PGC-1protein can readily be identified as comprising an amino acid sequencethat is substantially homologous to SEQ ID NO:7, 10, 13, or 16.

[0067] Moreover, nucleic acid molecules encoding other PGC-1 familymembers and, thus, which have a nucleotide sequence which differs fromthe PGC-1 sequences of SEQ ID NO:6, 8, 9, 11, 12, 14, 15, or 17, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number ______ or ______ are intended to be within the scopeof the invention. For example, another PGC-1 cDNA can be identifiedbased on the nucleotide sequence of human PGC-1. Moreover, nucleic acidmolecules encoding PGC-1 proteins from different species, and which,thus, have a nucleotide sequence which differs from the PGC-1 sequencesof SEQ ID NO:6, 8, 9,11, 12, 14, 15, or 17, or the nucleotide sequenceof the DNA insert of the plasmid deposited with ATCC as Accession Number______ or ______ are intended to be within the scope of the invention.For example, a mouse or monkey PGC-1 cDNA can be identified based on thenucleotide sequence of a human PGC-1.

[0068] Nucleic acid molecules corresponding to natural allelic variantsand homologues of the PGC-1 cDNAs of the invention can be isolated basedon their homology to the PGC-1 nucleic acids disclosed herein using thecDNAs disclosed herein, or a portion thereof, as a hybridization probeaccording to standard hybridization techniques under stringenthybridization conditions. Nucleic acid molecules corresponding tonatural allelic variants and homologues of the PGC-1 cDNAs of theinvention can further be isolated by mapping to the same chromosome orlocus as the PGC-1 gene.

[0069] Orthologues, homologues and allelic variants can be identifiedusing methods known in the art (e.g., by hybridization to an isolatednucleic acid molecule of the present invention, for example, understringent hybridization conditions). In one embodiment, an isolatednucleic acid molecule of the invention is at least 15, 20, 25, 30 ormore nucleotides in length and hybridizes under stringent conditions tothe nucleic acid molecule comprising the nucleotide sequence of SEQ IDNO:6, 8, 9, 11, 12, 14, 15, or 17, or the nucleotide sequence of the DNAinsert of the plasmid deposited with ATCC as Accession Number ______ or______. In other embodiment, the nucleic acid is at least 25, 26, 27,28, 30, 30, 36, 38, 40, 50, 59, 61, 87, 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 704, 750, 800, 843, 850, 873, 874,875, 876, 877, 900, 904, 905, 906, 906, 907, 908, 909, 910, 911, 924,925, 926, 927, 932, 933, 934, 939, 940, 941, 947, 948, 949, 950, 1000,1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700,1750, 1800, 1850 or more nucleotides in length.

[0070] As used herein, the term “hybridizes under stringent conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences that are significantly identical orhomologous to each other remain hybridized to each other. Preferably,the conditions are such that sequences at least about 70%, morepreferably at least about 80%, even more preferably at least about 85%or 90% identical to each other remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, Ausubel et al., eds.,John Wiley & Sons, Inc. (1995), sections 2, 4 and 6. Additionalstringent conditions can be found in Molecular Cloning: A LaboratoryManual, Sambrook et al., Cold. Spring Harbor Press, Cold Spring Harbor,N.Y. (1989), chapters 7, 9 and 11. A preferred, non-limiting example ofstringent hybridization conditions includes hybridization in 4× or 6×sodium chloride/sodium citrate (SSC), at about 65-70° C. (orhybridization in 4×SSC plus 50% formamide at about 42-50° C.) followedby one or more washes in 1×SSC, at about 65-70° C. A further preferred,non-limiting example of stringent hybridization conditions includeshybridization at 6×SSC at 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 65° C. A preferred, non-limiting example of highlystringent hybridization conditions includes hybridization in 1×SSC, atabout 65-70° C. (or hybridization in 1×SSC plus 50% formamide at about42-50° C.) followed by one or more washes in 0.3×SSC, at about 65-70° C.A preferred, non-limiting example of reduced stringency hybridizationconditions includes hybridization in 4×or 6×SSC, at about 50-60° C. (oralternatively hybridization in 6×SSC plus 50% fonnamide at about 40-45°C.) followed by one or more washes in 2×SSC, at about 50-60° C. Rangesintermediate to the above-recited values, e.g., at 65-70° C. or at42-50° C. are also intended to be encompassed by the present invention.SSPE (1×SSPE is 0.15 M NaCl, 10 mM NaH₂PO₄, and 1.25 mM EDTA, pH 7.4)can be substituted for SSC (1×SSC is 0.15 M NaCl and 15 mM sodiumcitrate) in the hybridization and wash buffers; washes are performed for15 minutes each after hybridization is complete. The hybridizationtemperature for hybrids anticipated to be less than 50 base pairs inlength should be 5-10° C. less than the melting temperature (T_(m)) ofthe hybrid, where T_(m) is determined according to the followingequations. For hybrids less than 18 base pairs in length, T_(m)(°C.)=2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and 49base pairs in length, T_(m)(° C.)=81.5+16.6(log₁₀[Na⁺])+0.41(%G+C)−(600/N), where N is the number of bases in the hybrid, and [Na⁺] isthe concentration of sodium ions in the hybridization buffer ([Na⁺] for1×SSC=0.165 M). It will also be recognized by the skilled practitionerthat additional reagents may be added to hybridization and/or washbuffers to decrease non-specific hybridization of nucleic acid moleculesto membranes, for example, nitrocellulose or nylon membranes, includingbut not limited to blocking agents (e.g., BSA or salmon or herring spermcarrier DNA), detergents (e.g., SDS), chelating agents (e.g., EDTA),Ficoll, PVP and the like. When using nylon membranes, in particular, anadditional preferred, non-limiting example of stringent hybridizationconditions is hybridization in 0.25-0.5M NaH₂PO₄, 7% SDS at about 65°C., followed by one or more washes at 0.02M NaH₂PO₄, 1% SDS at 65° C.,see e.g., Church and Gilbert (1984) Proc. Natl. Acad. Sci. USA81:1991-1995 (or alternatively 0.2×SSC, 1% SDS).

[0071] Preferably, an isolated nucleic acid molecule of the inventionthat hybridizes under stringent conditions to the sequence of SEQ IDNO:6, 8, 9, 11, 12, 14, 15, or 17 corresponds to a naturally-occurringnucleic acid molecule. As used herein, a “naturally-occurring” nucleicacid molecule refers to an RNA or DNA molecule having a nucleotidesequence that occurs in nature (e.g., encodes a natural protein).

[0072] In addition to naturally-occurring allelic variants of the PGC-1sequences that may exist in the population, the skilled artisan willfurther appreciate that changes can be introduced by mutation into thenucleotide sequences of SEQ ID NO:6, 8, 9, 11, 12, 14, 15, or 17, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number ______ or ______, thereby leading to changes in theamino acid sequence of the encoded PGC-1 proteins, without altering thefunctional ability of the PGC-1 proteins. For example, nucleotidesubstitutions leading to amino acid substitutions at “non-essential”amino acid residues can be made in the sequence of SEQ ID NO:6, 8, 9,11, 12, 14, 15, or 17, or the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number ______ or ______. A“non-essential” amino acid residue is a residue that can be altered fromthe wild-type sequence of PGC-1 (e.g., the sequence of SEQ ID NO:7, 10,13, or 16) without altering the biological activity, whereas an“essential” amino acid residue is required for biological activity. Forexample, amino acid residues that are conserved among the PGC-1 proteinsof the present invention, e.g., those present in a peroxisomallocalization signal, are predicted to be particularly unamenable toalteration. Furthermore, additional amino acid residues that areconserved between the PGC-1 proteins of the present invention and othermembers of the PGC-1 family are not likely to be amenable to alteration.

[0073] Accordingly, another aspect of the invention pertains to nucleicacid molecules encoding PGC-1 proteins that contain changes in aminoacid residues that are not essential for activity. Such PGC-1 proteinsdiffer in amino acid sequence from SEQ ID NO:7, 10, 13, or 16, yetretain biological activity. In one embodiment, the isolated nucleic acidmolecule comprises a nucleotide sequence encoding a protein, wherein theprotein comprises an amino acid sequence at least about 60%, 65%, 70%,75%, 80%, 85%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.25%, 99.75% or more homologous to SEQ ID NO:7, 10, 13, or 16, e.g.,to the entire length of SEQ ID NO:7, 10, 13, or 16.

[0074] An isolated nucleic acid molecule encoding a PGC-1 proteinhomologous to the protein of SEQ ID NO:7, 10, 13, or 16 can be createdby introducing one or more nucleotide substitutions, additions ordeletions into the nuclootide sequence of SEQ ID NO:6, 8, 9, 11, 12, 14,15, or 17, or the nucleotide sequence of the DNA insert oftheplasmid-deposited with ATCC as Accession Number ______ or ______, suchthat one or more amino acid substitutions, additions or deletions areintroduced into the encoded protein. Mutations can be introduced intoSEQ ID NO:6, 8, 9, 11, 12, 14, 15, or 17, or the nucleotide sequence ofthe DNA insert of the plasmid deposited with ATCC as Accession Number______ or ______ by standard techniques, such as site-directedmutagenesis and PCR-mediated mutagenesis. Preferably, conservative aminoacid substitutions are made at one or more predicted nonessential aminoacid residues. A “Conservative amino acid substitution” is one in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a PGC-1 protein ispreferably replaced with another amino acid residue from the same sidechain family. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of a PGC-1 coding sequence, suchas by saturation mutagenesis, and the resultant mutants can be screenedfor PGC-1 biological activity to identify mutants that retain activity.Following mutagenesis of SEQ ID NO:6, 8, 9, 11, 12, 14, 15, or 17, orthe nucleotide sequence of the DNA insert of the plasmid deposited withATCC as Accession Number ______ or ______, the encoded protein can beexpressed recombinantly and the activity of the protein can bedetermined.

[0075] In a preferred embodiment, a mutant PGC-1 protein can be assayedfor the ability to (i) interact with a PGC-1 target molecule; (i)modulate intracellular signaling; (iii) modulate cellular metabolism;(iv) localize to peroxisomes; (v) modulate the expression of genesinvolved in fatty acid uptake and/or oxidation (e.g., LPL, FAT/CD36,VLACS, AOX, MCAD, and/or MCD); (vi) modulate fatty acid uptake and/oroxidation; (vii) modulate energy homeostasis; and/or (viii) modulatelipid homeostasis.

[0076] In addition to the nucleic acid molecules encoding PGC-1 proteinsdescribed above, another aspect of the invention pertains to isolatednucleic acid molecules which are antisense thereto. In an exemplaryembodiment, the invention provides an isolated nucleic acid moleculewhich is antisense to a PGC-1 nucleic acid molecule (e.g., is antisenseto the coding strand of a PGC-1 nucleic acid molecule). An “antisense”nucleic acid comprises a nucleotide sequence which is complementary to a“sense” nucleic acid encoding a protein, e.g., complementary to thecoding-strand of a double-stranded cDNA molecule or complementary to anmRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bondto a sense nucleic acid. The antisense nucleic acid can be complementaryto an entire PGC-1 coding strand, or to only a portion thereof. In oneembodiment, an antisense nucleic acid molecule is antisense to “codingregion sequences” of the coding strand of a nucleotide sequence encodingPGC-1. The term “coding region sequences” refers to the region of thenucleotide sequence comprising codons which are translated into aminoacid residues (e.g., the coding region sequences of human PGC-1bcorresponding to SEQ ID NO:8, or the coding region sequences of humanPGC-1c corresponding to SEQ ID NO: 14). In another embodiment, theantisense nucleic acid molecule is antisense to a “noncoding region” ofthe coding strand of a nucleotide sequence encoding PGC-1. The term“noncoding region” refers to 5′ and/or 3′ sequences which flank thecoding region sequences that are not translated into amino acids (alsoreferred to as 5′ and 3′ untranslated regions).

[0077] Given the coding strand sequences encoding PGC-1 disclosed herein(e.g., SEQ ID NO:8 or 14), antisense nucleic acids of the invention canbe designed according to the rules of Watson and Crick base pairing. Theantisense nucleic acid molecule can be complementary to coding regionsequences of PGC-1 mRNA, but more preferably is an oligonucleotide whichis antisense to only a portion of the PGC-1 mRNA. An antisenseoligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length. Anantisense nucleic acid of the invention can be constructed usingchemical synthesis and enzymatic ligation reactions using proceduresknown in the art. For example, an antisense nucleic acid (e.g., anantisense oligonucleotide) can be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed between the antisense and sensenucleic acids, e.g., phosphorothioate derivatives and acridinesubstituted nucleotides can be used. Examples of modified nucleotideswhich can be used to generate the antisense nucleic acid include5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4acetylcytosine, 5-(carboxyhydroxylmethyl)uracil,5 carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-direthylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

[0078] The antisense nucleic acid molecules of the invention aretypically administered to a subject or generated in situ such that theyhybridize with or bind to cellular mRNA and/or genomic DNA encoding aPGC-1 protein to thereby inhibit expression of the protein, e.g., byinhibiting transcription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. An example of a route of administration of antisensenucleic acid molecules of the invention include direct injection at atissue site. Alternatively, antisense nucleic acid molecules can bemodified to target selected cells and then administered systemically.For example, for systemic administration, antisense molecules can bemodified such that they specifically bind to receptors or antigensexpressed on a selected cell surface, e.g. by linking the antisensenucleic acid molecules to peptides or antibodies which bind to cellsurface receptors or antigens. The antisense nucleic acid molecules canalso be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of the antisensemolecules, vector constructs in which the antisense nucleic acidmolecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

[0079] In yet another embodiment, the antisense nucleic acid molecule ofthe invention is an α-anomeric nucleic acid molecule. An α-anomericnucleic acid molecule forms specific double-stranded hybrids withcomplementary RNA in which, contrary to the usual β-units, the strandsrun parallel to each other (Gaultier et al. (1987) Nucleic Acids Res.15:6625-6641). The antisense nucleic acid molecule can also comprise a2′-o-methytribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

[0080] In still another embodiment, an antisense nucleic acid of theinvention is a ribozyme. Ribozymes are catalytic RNA molecules withribonuclease activity which are capable of cleaving a single-strandednucleic acid, such as an mRNA, to which they have a complementaryregion. Thus, ribozymes (e.g., hammerhead ribozymes (described inHaseloff and Gerlach (1988) Nature 334:585-591)) can be used tocatalytically cleave PGC-1 mRNA transcripts to thereby inhibittranslation of PGC-1 mRNA. A ribozyme having specificity for aPGC-1-encoding nucleic acid can be designed based upon the nucleotidesequence of a PGC-1 cDNA disclosed herein (i.e., SEQ ID NO:6, 8, 9, 11,12, 14, 15, or 17, or the nucleotide sequence of the DNA insert of theplasmid deposited with ATCC as Accession Number _____ or ______). Forexample, a derivative of a Tetrahymena L-19 IVS RNA can be constructedin which the nucleotide sequence of the active site is complementary tothe nucleotide sequence to be cleaved in a PGC-1-encoding mRNA. See,e.g. Cech et al., U.S. Pat. No. 4,987,071; and Cech et al., U.S. Pat.No. 5,116,742. Alternatively, PGC-1 mRNA can be used to select acatalytic RNA having a specific ribonuclease activity from a pool of RNAmolecules. See, e.g., Bartel, D. and Szostak, J. W. (1993) Science261:1411-1418.

[0081] Alternatively, PGC-1 gene expression can be inhibited bytargeting nucleotide sequences complementary to the regulatory region ofthe PGC-1 gene (e.g., the PGC-1b or PGC-1c promoters and/or enhancers;e.g., nucleotides 1-89 of SEQ ID NO:6 or nucleotides 1-66 of SEQ ID NO:12) to form triple helical structures that prevent transcription of thePGC-1 gene in target cells. In another embodiment, the sequences uniqueto PGC-1b or PGC-1c can be targeted (e.g, SEQ ID NO:9 or SEQ ID NO:15).See generally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84;Helene, C. et al. 1992) Ann. N.Y. Acad. Sci. 660:27-36;and Maher, L.J.(1992) Bioessays 14(12):807-15.

[0082] In yet another embodiment, the PGC-1 nucleic acid molecules ofthe present invention can be modified at the base moiety, sugar moietyor phosphate backbone to improve, e.g., the stability, hybridization, orsolubility of the molecule. For example, the deoxyribose phosphatebackbone of the nucleic acid molecules can be modified to generatepeptide nucleic acids (see Hyrup, B. and Nielsen, P. E. (1996) Bioorg.Med Chem. 4(1):5-23). As used herein, the terms “peptide nucleic acids”or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which thedeoxyribose phosphate backbone is replaced by a pseudopeptide backboneand only the four natural nucleobases are retained. The neutral backboneof PNAs has been shown to allow for specific hybridization to DNA andRNA under conditions of low ionic strength. The synthesis of PNAoligomers can be performed using standard solid phase peptide synthesisprotocols as described in Hyrup and Nielsen (1996) supra andPerry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93:14670-675.

[0083] PNAs of PGC-1 nucleic acid molecules can be used in therapeuticand diagnostic applications. For example, PNAs can be used as antisenseor antigene agents for sequence-specific modulation of gene expressionby, for example, inducing transcription or translation arrest orinhibiting replication. PNAs of PGC-1 nucleic acid molecules can also beused in the analysis of single base pair mutations in a gene (e.g., byPNA-directed PCR clamping); as ‘artificial restriction enzymes’ whenused in combination with other enzymes (e.g., S1 nucleases (Hyrup andNielsen (1996) supra)); or as probes or primers for DNA sequencing orhybridization (Hyrup and Nielsen (1996) supra; Perry-O'Keefe et al.(1996) supra).

[0084] In another embodiment, PNAs of PGC-1 can be modified (e.g.,.toenhance their stability or cellular uptake), by attaching lipophilic orother helper groups to PNA, by *the formation of PNA-DNA chimeras, or bythe use of liposomes or other techniques of drug delivery known in theart. For example, PNA-DNA chimeras of PGC-1 nucleic acid molecules canbe generated which may combine the advantageous properties of PNA andDNA. Such chimeras allow DNA recognition enzymes (e.g., RNase H and DNApolymerases) to interact with the DNA portion while the PNA portionwould provide high binding affinity and specificity. PNA-DNA chimerascan be linked using linkers of appropriate lengths selected in terms ofbase stacking, number of bonds between the nucleobases, and orientation(Hyrup and Nielsen (1996) supra). The synthesis of PNA-DNA chimeras canbe performed as described in Hyrup and Nielsen (1996) supra and Finn, P.J. et al. (1996) Nucleic Acids Res. 24(17):3357-63. For example, a DNAchain can be synthesized on a solid support using standardphosphoramidite coupling chemistry and modified nucleoside analogs,e.g., 5′-(4 -methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, canbe used as a between the PNA and the 5′ end of DNA (Mag, M. et al.(1989) Nucleic Acids Res. 17:5973-88). PNA monomers are then coupled ina stepwise manner to produce a chimeric molecule with a 5′ PNA segmentand a 3′ DNA segment (Finn et al. (1996) supra). Alternatively, chimericmolecules can be synthesized with a 5′ DNA segment and a 3′ PNA segment(Peterser, K. H. et al. (1975) Bioorganic Med Chem. Lett. 5:1119-11124).

[0085] In other embodiments, the oligonucleotide may include otherappended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad Sci. USA86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA84:648-652; PCT Publication No. WO 88/09810) or the blood-brain barrier(see, e.g., PCT Publication No. WO 89/10134). In addition,oligonucleotides can be modified with hybridization-triggered cleavageagents (See, e.g., Krol et al. (1988) Biotechniques 6:958-976) orintercalating agents (See, e.g., Zon (1988) Pharm. Res. 5:539-549). Tothis end, the oligonucleotide may be conjugated to another molecule(e.g., a peptide, hybridization triggered cross-linking agent, transportagent, or hybridization-triggered cleavage agent).

[0086] II. Isolated PGC-1 Proteins and Anti-PGC-1 Antibodies

[0087] One aspect of the invention pertains to isolated or recombinantPGC-1 proteins and polypeptides, and biologically active portionsthereof, as well as polypeptide fragments suitable for use as immunogensto raise anti-PGC-1 antibodies. In one embodiment, native PGC-1 proteinscan be isolated from cells or tissue sources by an appropriatepurification scheme using standard protein purification techniques. Inanother embodiment, PGC-1 proteins are produced by recombinant DNAtechniques. Alternative to recombinant expression, a PGC-1 protein orpolypeptide can be synthesized chemically using standard peptidesynthesis techniques.

[0088] An “isolated” or “purified” protein or biologically activeportion thereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which thePGC-1 protein is derived, or substantially free from chemical precursorsor other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations of PGC-1protein in which the protein is separated from cellular components ofthe cells from which it is isolated or recombinantly produced. In oneembodiment, the language “substantially free of cellular material”includes preparations of PGC-1 protein having less than about 30% (bydry weight) of non-PGC-1 protein (also referred to herein as a“contaminating protein”), more preferably less than about 20% ofnon-PGC-1 protein, still more preferably less than about 10% ofnon-PGC-1 protein, and most preferably less than about 5% non-PGC-1protein. When the PGC-1 protein or biologically active portion thereofis recombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,more preferably less than about 10%, and most preferably less than about5% of the volume of the protein preparation.

[0089] The language “substantially free of chemical precursors or otherchemicals” includes preparations of PGC-1 protein in which the proteinis separated from chemical precursors or other chemicals which areinvolved in the synthesis of the protein. In one embodiment, thelanguage “substantially free of chemical precursors or other chemicals”includes preparations of PGC-1 protein having less than about 30% (bydryweight) of chemical precursors or non-PGC-1 chemicals, more preferablyless than about 20% chemical precursors or non-PGC-1 chemicals, stillmore preferably less than about 10% chemical precursors or non-PGC-1chemicals, and most preferably less than about 5% chemical precursors ornon-PGC-1 chemicals.

[0090] As used herein, a “biologically active portion” of a PGC-1protein includes a fragment of a PGC-1 protein which participates in aninteraction between a PGC-1 molecule and a non-PGC-1 molecule or whichis capable of modulating expression of genes involved in fatty aciduptake and/or oxidation. Biologically active portions of a PGC-1 proteininclude peptides comprising amino acid sequences sufficiently homologousto or derived from the PGC-1 amino acid sequences, e.g. the amino acidsequences shown in SEQ ID NO:7, 10, 13, or 16, which include sufficientamino acid residues to exhibit at least one activity of a PGC-1 protein.Typically, biologically active portions comprise a domain or motif withat least one activity of the PGC-1 protein, e.g., modulation of theexpression of genes involved in fatty acid uptake and/or oxidation. Abiologically active portion of a PGC-1 protein can be a polypeptidewhich is, for example, 9, 10, 25, 28, 29, 30, 31, 35, 40, 45, 50, 75,100, 125, 150, 175, 200, 225, 250, 291, 292, 293, 294, 295, 300 or moreamino acids in length. Biologically active portions of a PGC-1 proteincan be used as targets for developing agents which modulate a PGC-1mediated activity, e.g., modulation of the expression of genes involvedin fatty acid uptake and/or oxidation.

[0091] In one embodiment, a biologically active portion of a PGC-1protein comprises at least one tyrosine phosphorylation motif, one cAMPphosphorylation motif, one LXXLL motif, and/or one peroxisomallocalization signal. Moreover, other biologically active portions, inwhich other regions of the protein are deleted, can be prepared byrecombinant techniques and evaluated for one or more of the functionalactivities of a native PGC-1 protein.

[0092] Another aspect of the invention features fragments of the proteinhaving the amino acid sequence of SEQ ID NO:7, 10, 13, or 16, forexample, for use as immunogens. In one embodiment, a fragment comprisesat least 8 amino acids (e.g., contiguous or consecutive amino acids) ofthe amino acid sequence of SEQ ID NO:7, 10, 13, or 16, or an amino acidsequence encoded by the DNA insert of the plasmid deposited with theATCC as Accession Number _____ or ______. In another embodiment, afragment comprises at least 8, 9, 10, 15, 20, 25, 28, 29, 30, 31, 35,40, 45, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 291, 292, 293,294, 295, 300 or more amino acids (e.g., contiguous or consecutive aminoacids) of the amino acid sequence of SEQ ID NO:7, 10, 13, or 16, or anamino acid sequence encoded by the DNA insert of the plasmid depositedwith the ATCC as Accession Number ______ or ______.

[0093] In another embodiment, a fragment comprises at least 8, 9, 10,15, 20, 25, 28, 29, 30, 31, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200,225, 250, 275, 291, 292, 293, 25 294, 295, 300 or more contiguous aminoacid residues of SEQ ID NO:7 and includes at least residue 292 of SEQ IDNO:7. In still another embodiment, a fragment comprises at least 8, 9,10, 15, 20, 25, 28, 29, 30, 31, 35, 40, 45, 50, 75, 100, 125, 150, 175,200, 225, 250, 275, 291, 292, 293, 294, 295 or more contiguous aminoacid residues of SEQ ID NO:13 and includes at least residue 293of SEQ IDNO:13.

[0094] In a preferred embodiment, a PGC-1 protein has an amino acidsequence shown in SEQ ID NO:7, 10, 13, or 16. In other embodiments, thePGC-1 protein is substantially identical to SEQ ID NO:7, 10, 13, or 16,and retains the functional activity of the protein of SEQ ID NO:7, 10,13, or 16, yet differs in amino acid sequence due to natural allelicvariation or mutagenesis, as described in detail in subsection I above.In another embodiment, the PGC-1 protein is a protein which comprises anamino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.25%, 99.5%, 99.75%or more identical to SEQ ID NO:7, 10, 13, or 16.

[0095] In another embodiment, the invention features a PGC-1 proteinwhich is encoded by a nucleic acid molecule consisting of a nucleotidesequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.25%, 99.5%, 99.75% or moreidentical to a nucleotide sequence of SEQ ID NO:6, 8, 9, 11, 12, 14, 15,or 17, or a complement thereof. This invention futrther features a PGC-1protein which is encoded by a. nucleic acid molecule consisting of anucleotide sequence which hybridizes under stringent hybridizationconditions to a complement of a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:6, 8, 9, 11, 12, 14, 15, or 17, or acomplement thereof.

[0096] To determine the percent identity of two amino acid sequences orof two nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). In a preferred embodiment, the lengthof a reference sequencealigned for comparison purposes isat least 30%; preferably at least 40%,more preferably at least 50%, even more preferably at least 60%, andeven more preferably at least 70%, 80%, or 90%. of the length of thereference sequence (eg.g when aligning a second sequence to the PGC-1bamino acid sequence of SEQ ID NO:7 having 320 amino acid residues, atleast 96, preferablyat least 128, more preferably at least 160, evenmore preferably at least .192, and even more preferably at least 224,256 or 288 amino acid residues are aligned; when aligning asecond.sequence to the PGC-1c amino acid sequence of SEQ ID NO:13 having300 amino acid residues at least 90 preferably at least 120, morepreferably at least 150, even more preferably atleast 180, and even morepreferably at least 210, 240 or 270 amino acid residues are aligned).The amino acid residues or nucleotides at corresponding amino acidpositions or nucleotide ositions are then compared. When a position inthe first sequence is occupied by the same amino acid residue ornucleotide as the corresponding position in the second sequence, thenthe molecules are identical at that position (as used herein amino acidor nucleic acid “identity” is equivalent to amino acid or nucleic acid“homology”). The percent identity between the two sequences is afunction of the number of identical positions shared by the sequences,taking into account the number of gaps, and the length of each gap,which need to be introduced for optimal alignment of the two sequences.

[0097] The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch (J.Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporatedinto the GAP program in the GCG software package (available onlinethrough the Genetics Computer Group) using either a Blossum 62 matrix ora PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and alength weight of 1, 2, 3, 4, 5, or 6. In yet another preferredembodiment, the percent identity between two nucleotide sequences isdetermined using the GAP program in the GCG software package (availableat (available online through the Genetics Computer Group), using aNWSgapdna. CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and alength weight of 1, 2, 3, 4, 5, or 6. A preferred, non-limiting exampleof parameters to be used in conjunction with the GAP program include aBlossum 62 scoring matrix with a gap penalty of 12, a gap extend penaltyof 4, and a frame shift gap penalty of 5.

[0098] In another embodiment, the percent identity between two aminoacid or nucleotide sequences is determined using the algorithm of Meyersand Miller (Comput. Appl. Biosci. 4:11-17 (1988)) which has beenincorporated into the ALIGN program (version 2.0 or version 2.0U), usinga PAM120 weight residue table, a gap length penalty of 12 and a gappenalty of 4.

[0099] The nucleic acid and protein sequences of the present inventioncan further be used as a “query sequence” to perform a search againstpublic databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to PGC-1 nucleic acid molecules of the invention. BLASTprotein searches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to PGC-1 proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.(1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST can be used. See the website for the NationalCenter for Biotechnology Information.

[0100] The invention also provides PGC-1 chimeric or fusion proteins. Asused herein, a PGC-1 “chimeric protein” or “fusion protein” comprises aPGC-1 polypeptide operatively linked to a non-PGC-1 polypeptide. A“PGC-1 polypeptide” refers to a polypeptide having an amino acidsequence corresponding to PGC-1 (e.g., PGC-1 or PGC-1), whereas a“non-PGC-1 polypeptide” refers to a polypeptide having an amino acidsequence corresponding to a protein which is not substantiallyhomologous to the PGC-1 protein, e.g., a protein which is different fromthe PGC-1 protein and which is derived from the same or a differentorganism Within a PGC-1 fusion protein the PGC-1 polypeptide cancorrespond to all or a portion of a PGC-1 protein. In a preferredembodiment, a PGC-1 fusion protein comprises at least one biologicallyactive portion of a PGC-1 protein. In another preferred embodiment, aPGC-1 fusion protein comprises at least two biologically active portionsof a PGC-1 protein. Within the fusion protein, the term “operativelylinked” is intended to indicate that the PGC-1 polypeptide and thenon-PGC-1 polypeptide are fused in frame to each other. The non-PGC-1polypeptide can be fused to the N-terminus or C-terminus of the PGC-1polypeptide.

[0101] For example, in one embodiment, the fusion protein is a GST-PGC-1fusion protein in which the PGC-1 sequences are fused to the C-terminusof the GST sequences. Such fusion proteins can facilitate thepurification of recombinant PGC-1. In another embodiment, the fusionprotein is a PGC-1 protein containing a heterologous signal sequence atits N-terminus. In certain host cells (e.g., mammalian host cells),expression and/or secretion of PGC-1 can be increased through use of aheterologous signal sequence. In another embodiment the fusion proteinis a GFP-PGC-1 fusion protein in which the PGC-1 sequences are fused tothe green fluorescent protein (GFP), which allows visualization of thePGC-1 fusion protein in live cells.

[0102] The PGC-1 fusion proteins of the invention can be incorporatedinto pharmaceutical compositions and administered to a subject in vivo.The PGC-1 fusion proteins can be used to affect the bioavailability of aPGC-1 target molecule. Use of PGC-1 fusion proteins may be usefultherapeutically for the treatment of disorders caused by, for example,(i) aberrant modification or mutation of a gene encoding a PGC-1protein; (ii) mis-regulation of the PGC-1 gene; and (iii) aberrantpost-translational modification of a PGC-1 protein.

[0103] Moreover, the PGC-1-fusion proteins of the invention can be usedas immunogens to produce anti-PGC-1 antibodies in a subject, to purifyPGC-1 target molecules, and in screening assays to identify moleculeswhich inhibit or enhance the transport of a PGC-1 substrate or theinteraction of PGC-1 with a PGC-1 target molecule.

[0104] Preferably, a PGC-1 chimeric or fusion protein of the inventionis produced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and reamplified to generatea chimeric gene sequence (see, for example, Current Protocols inMolecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST or GFP polypeptide). APGC-1-encoding nucleic acid can be cloned into such an expression vectorsuch that the fusion moiety is linked in-frame to the PGC-1 protein.

[0105] The present invention also pertains to variants of the PGC-1proteins which function as either PGC-1 agonists (mimetics) or as PGC-1antagonists. Variants of the PGC-1 proteins can be generated bymutagenesis, e.g., discrete point mutation or truncation of a PGC-1protein. An agonist of the PGC-1 proteins can retain substantially thesame, or a subset, of the biological activities of the naturallyoccurring form of a PGC-1 protein. An antagonist of a PGC-1 protein caninhibit one or more of the activities of the naturally occurring form ofthe PGC-1 protein by, for example, competitively modulating aPGC-1-mediated activity of a PGC-1 protein. Thus, specific biologicaleffects can be elicited by treatment with a variant of limited function.In one embodiment, treatment of a subject with a variant having a subsetof the biological activities of the naturally occurring form of theprotein has fewer side effects in a subject relative to treatment withthe naturally occurring form of the PGC-1 protein.

[0106] In one embodiment, variants of a PGC-1 protein which function aseither PGC-1 agonists (mimetics) or as PGC-1 antagonists can beidentified by screening combinatorial libraries of mutants, e.g.,truncation mutants, of a PGC-1 protein for PGC-1 protein agonist orantagonist activity. In one embodiment, a variegated library of PGC-1variants is generated by combinatorial mutagenesis at the nucleic acidlevel and is encoded by a variegated gene library. A variegated libraryof PGC-1 variants can be produced by, for example, enzymaticallyligating a mixture of synthetic oligonucleotides into gene sequencessuch that a degenerate set of potential PGC-1 sequences is expressibleas individual polypeptides, or alternatively, as a set of larger fusionproteins (e.g., for phage display) containing the set of PGC-1-sequencestherein. There are a variety of methods which can be used to producelibraries of potential PGC-1 variants from a degenerate oligonucleotidesequence. Chemical synthesis of a degenerate gene sequence can beperformed in an automatic DNA synthesizer, and the synthetic gene thenligated into an appropriate expression vector. Use of a degenerate setof genes allows for the provision, in one mixture, of all of thesequences encoding the desired set of potential PGC-1 sequences. Methodsfor synthesizing degenerate oligonucleotides are known in the art (see,e.g.,.Narang, S. A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu.Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al.(1983) Nucleic. Acids Res. 11:477).

[0107] In addition, libraries of fragments of a PGC-1 protein codingsequence can be used to generate a variegated population of PGC-1fragments for screening and subsequent selection of variants of a PGC-1protein. In one embodiment, a library of coding sequence fragments canbe generated by treating a double stranded PCR fragment of a PGC-1coding sequence with a nuclease under conditions wherein nicking occursonly about once per molecule, denaturing the double stranded DNA,renaturing the DNA to form double stranded DNA which can includesense/antisense pairs from different nicked products removing singlestranded portions from reformed duplexes by treatment with S1 nuclease,and ligating the resulting fragment library into an expression vector.By this method, an expression library can be derived which encodesN-terminal, C-terminal and internal fragments of various sizes of thePGC-1 protein.

[0108] Several techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations ortruncation, and for screening cDNA libraries for gene products having aselected property. Such techniques are adaptable for rapid screening ofthe gene libraries generated by the combinatorial mutagenesis of PGC-1proteins. The most widely used techniques, which are amenable to highthrough-put analysis, for screening large gene libraries typicallyinclude cloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates isolation of the vectorencoding the gene whose product was detected. Recursive ensemblemutagenesis (REM), a new technique which enhances the frequency offunctional mutants in the libraries, can be used in combination with thescreening assays to identify PGC-1 variants (Arkin and Youvan (1992)Proc. Natl. Acad. Sci. USA 89:7811-7815; Delagrave et al. (1993) ProteinEng. 6(3):327-331).

[0109] In one embodiment, cell based assays can be exploited to analyzea variegated PGC-1 library. For example, a library of expression vectorscan be transfected into a cell line which ordinarily responds to PGC-1in a particular PGC-1 dependent manner. The transfected cells are thencontacted with PGC-1 and the effect of the expression of the mutant onsignaling by the PGC-1 can be detected, e.g, by measuring expression ofgenes involved in fatty acid uptake and/or oxidation. Plasmid DNA canthen be recovered from the cells which score for inhibition, oralternatively, potentiation of signaling by the PGC-1 mutant, and theindividual clones further characterized.

[0110] An isolated PGC-1 protein, or a portion or fragment thereof, canbe used as an immunogen to generate antibodies that bind PGC-1 usingstandard techniques for polyclonal and monoclonal antibody preparation.A full-length PGC-1 protein can be used or, alternatively, the inventionprovides antigenic peptide fragments of PGC-1 for use as immunogens. Theantigenic peptide of PGC-1 comprises at least 8 amino acid residues ofthe amino acid sequence shown in SEQ ID NO:7, 10, 13, or 16 andencompasses an epitope of PGC-1 such that an antibody raised against thepeptide forms a specific immune complex with PGC-1. Preferably, theantigenic peptide comprises at least 10 amino acid residues, morepreferably at least 15 amino acid residues, even more preferably atleast 20 amino acid residues, and most preferably at least 30 amino acidresidues. In another preferred embodiment, the antigenic peptidecomprises an amino acid sequence unique to PGC-1b or PGC-1c, forexample, a peptide comprising at least 8 amino acid residues of theamino acid sequence shown in SEQ ID NO:10 or 16.

[0111] Preferred epitopes encompassed by the antigenic peptide areregions of PGC-1 that are located on the surface of the protein, e.g.,hydrophilic regions, as well as regions with high antigenicity.

[0112] A PGC-1 immunogen typically is used to prepare antibodies byimmunizing a suitable subject (e.g., rabbit, goat, mouse, or othermammal) with the immunogen. An appropriate immunogenic preparation cancontain, for example, recombinantly expressed PGC-1 protein or achemically-synthesized PGC-1 polypeptide. The preparation can furtherinclude an adjuvant, such as Freund's complete or incomplete adjuvant,or similar immunostimulatory agent Immunization of a suitable subjectwith an immunogenic PGC-1 preparation induces a polyclonal anti-PGC-1antibody response.

[0113] Accordingly, another aspect of the invention pertains toanti-PGC-1 antibodies. The term “antibody” as used herein refers toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site which specifically binds (immunoreacts with) an antigen,such as PGC-1. Examples of immunologically active portions ofimmunoglobulin molecules include F(ab) and F(ab′)₂ fragments which canbe generated by treating the antibody with an enzyme such as pepsin. Theinvention provides polyclonal and monoclonal antibodies that bind PGC-1.The term “monoclonal antibody” or “monoclonal antibody composition”, asused herein, refers to a population of antibody molecules that containonly one species of an antigen binding site capable of immunoreactingwith a particular epitope of PGC-1. A monoclonal antibody compositionthus typically displays a single binding affinity for a particular PGC-1protein with which it immunoreacts.

[0114] Polyclonal anti-PGC-1 antibodies can be prepared as describedabove by immunizing a suitable subject with a PGC-1 immunogen. Theanti-PGC-1 antibody titer in the immunized subject can be monitored overtime by standard techniques, such as with an enzyme linked immunosorbentassay (ELISA) using immobilized PGC-1. If desired, the antibodymolecules directed against PGC-1 can be isolated from the mammal (e.g.,from the blood) and further purified by well known techniques, such asprotein A chromatography to obtain the IgG fraction. At an appropriatetime after immunization, e.g., when the anti-PGC-1 antibody titers arehighest, antibody-producing cells can be obtained from the subject andused to prepare monoclonal antibodies by standard techniques, such asthe hybridoma technique originally described by Kohler and Mulstein(1975) Nature 256:495-497 (see also Brown et al. (1981) J. Immunol.127:539-46; Brown et al. (1980) J. Biol. Chem. 255:4980-83; Yeh et al.(1976) Proc. Natl. Acad. Sci. USA 76:2927-31; and Yeh et al. (1 982)Int. J. Cancer 29:269-75), the more recent human B cell hybridomatechnique (Kozbor et al. (1983) Immunol. Today 4:72), the EBV-hybridomatechnique (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy,Alan R Liss, Inc., pp. 77-96) or trioma techniques. The technology forproducing monoclonal antibody hybridomas is well known (see generallyKenneth, R H. in Monoclonal Antibodies: A New Dimension In BiologicalAnalyses, Plenum Publishing Corp., New York, N.Y. (1980); Lemer, E. A.(1981) Yale J. Biol. Med 54:387402; Gefter, M. L. et al. (1977) SomaticCell Genet. 3:231-36). Briefly, an immortal cell line (typically amyeloma) is fused to lymphocytes (typically splenocytes) from a mammalimmunized with a PGC-1 immunogen as described above, and the culturesupernatants of the resulting hybridoma cells are screened to identify ahybridoma producing a monoclonal antibody that binds PGC-1.

[0115] Any of the many well known protocols used for fusing lymphocytesand immortalized cell lines can be applied for the purpose of generatingan anti-PGC-1 monoclonal antibody (see, e.g., Galfre, G. et al. (1977)Nature 266:55052; Gefter et al. (1997) supra; Lerner (1981) supra;Kenneth (1980) supra). Moreover, the ordinarily skilled worker willappreciate that there are many variations of such methods which alsowould be useful. Typically, the immortal cell line (e.g., a myeloma cellline) is derived from the same mammalian species as the lymphocytes. Forexample, murine hybridomas can be made by fusing lymphocytes from amouse immunized with an immunogenic preparation of the present inventionwith an immortalized mouse cell line. Preferred immortal cell lines aremouse myeloma cell lines that are sensitive to culture medium containinghypoxanthine, aminopterin and thymidine (“HAT medium”). Any of a numberof myeloma cell lines can be used as a fusion partner according tostandard techniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 orSp2/O-Ag14 myeloma lines. These myeloma lines are available from ATCC.Typically, HAT-sensitive mouse myeloma cells are fused to mousesplenocytes using polyethylene glycol (“PEG”). Hybridoma cells resultingfrom the fusion are then selected using HAT medium, which kills unfusedand unproductively fused myeloma cells (unfused splenocytes die afterseveral days because they are not transformed). Hybridoma cellsproducing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bindPGC-1, e.g., using a standard ELISA assay.

[0116] Alternative to preparing monoclonal antibody-secretinghybridomas, a monoclonal anti-PGC-1 antibody can be identified andisolated by screening a recombinant combinatorial immunoglobulin library(e.g. an antibody phage display library) with PGC-1 to thereby isolateimmunoglobulin library members that bind PGC-1. Kits for generating andscreening phage display libraries are commercially available (e.g., thePharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; andthe Stratagene SurfZAP™ Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, Ladner et al., U.S. Pat. No. 5,223,409; Kang et al.,PCT International Publication No. WO 92/18619; Dower et al., PCTInternational Publication No. WO 91/17271; Winteret al., PCTInternational Publication No. WO 92/20791; Markland et al., PCTInternational Publication No. WO 92/15679; Breitling et al., PCTInternational Publication No. WO 93/01288; McCafferty et al., PCTInternational Publication No. WO 92/01047; Garrard et al., PCTInternational Publication No. WO 92/09690; Ladner et al., PCTInternational Publication. No. WO 90/02809; Fuchs et al. (1991)Biotechnology (NY) 9:1369-1372; Hay et al. (1992) Hum. Antibod.Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffithset al. (1993) EMBO J. 12:725-734; Hawkins et al. (1992) J. Mol. Biol.226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al.(1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrard et al. (1991)Biotechnology (NY) 9:1373-1377; Hoogenboom et al. (1991) Nucleic AcidsRes. 19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA88:7978-7982; and McCafferty et al. (1990) Nature 348:552-554.

[0117] Additionally, recombinant anti-PGC-1 antibodies, such as chimericand humanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in Robinson etal., International Application No. PCT/US86/02269; Akira et al.,European Patent Application No. 184,187; Taniguchi, M., European PatentApplication No. 171,496; Morrison et al., European Patent ApplicationNo. 173,494; Neuberger et al., PCT International Publication No. WO86/01533; Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al.,European Patent Application No. 125,023; Better et al. (1988) Science240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al.(1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987)Cancer Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shawet al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, S. L.(1985) Science 229:1202-1207; Oi et al. (1986) Biotechniques 4:214;Winter, U.S. Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-525;Verhoeyen et al. (1988) Science 239:1534; and Beidler et al. (1988) J.Immunol. 141:4053-4060.

[0118] An anti-PGC-1 antibody (e.g., monoclonal antibody) can be used toisolate PGC-1 by standard techniques, such as affinity chromatography orimmunoprecipitation. An anti-PGC-1 antibody can facilitate thepurification of natural PGC-1 from cells and of recombinantly producedPGC-1 expressed in host cells. Moreover, an anti-PGC-1 antibody can beused to detect PGC-1 protein (e.g., in a cellular lysate or cellsupernatant) in order to evaluate the abundance and pattern ofexpression of the PGC-1 protein. Anti-PGC-1 antibodies can be useddiagnostically to monitor protein levels in tissue as part of a clinicaltesting procedure, e.g., to, for example, determine the efficacy of agiven treatment regiment. Detection can be facilitated by coupling(i.e., physically linking) the antibody to a detectable substance.Examples of detectable substances include various enzymes, prostheticgroups, fluorescent materials, luminescent materials, bioluminescentmaterials, and radioactive materials. Examples of suitable enzymesinclude horseradish peroxidase, alkaline phosphatase, β-galactosidase,or acetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerytlrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

[0119] II. Recombinant Expression Vectors and Host Cells

[0120] Another aspect of the invention pertains to vectors, for examplerecombinant expression vectors, containing a PGC-1 nucleic acid moleculeor vectors containing a nucleic acid molecule which encodes a PGC-1protein (or a portion thereof). As used herein, the term “vector” refersto a nucleic acid molecule capable of transporting another nucleic acidto which it has been linked. One type of vector is a “plasmid”, whichrefers to a circular double stranded DNA loop into which additional DNAsegments can be ligated. Another type of vector is a viral vector,wherein additional DNA segments can be ligated into the viral genome.Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g., bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” can be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

[0121] The recombinant expression vectors of the invention comprise anucleic acid of the invention in a form suitable for expression of thenucleic acid in a host cell, which means that the recombinant expressionvectors include one or more regulatory sequences, selected on the basisof the host cells to be used for expression, which is operatively linkedto the nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory sequence(s)in a manner which allows for expression of the nucleotide sequence(e.g., in an in vitro transcription/translation system or in a host cellwhen the vector is introduced into the host cell). The term “regulatorysequence” is intended to include promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Suchregulatory sequences are described, for example, in Goeddel (1990)Methods Enzymol. 185:3-7. Regulatory sequences include those whichdirect constitutive expression of a nucleotide sequence in many types ofhost cells and those which direct expression of the nucleotide sequenceonly in certain host cells (e.g., tissue-specific regulatory sequences).It will be appreciated by those skilled in the art that the design ofthe expression vector can depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,and the like. The expression vectors of the invention can be introducedinto host cells to thereby produce proteins or peptides, includingfusion proteins or peptides, encoded by nucleic acids as describedherein (e.g., PGC-1 proteins, mutant forms of PGC-1 proteins, fusionproteins, and the like).

[0122] Accordingly, an exemplary embodiment provides a method forproducing a protein, preferably a PGC-1 protein, by culturing in asuitable medium a host cell of the invention (e.g., a mammalian hostcell such as a non-human mammalian cell) containing a recombinantexpression vector, such that the protein is produced.

[0123] The recombinant expression vectors of the invention can bedesigned for expression of PGC-1 proteins in prokaryotic or eukaryoticcells. For example, PGC-1 proteins can be expressed in bacterial cellssuch as E. coli, insect cells (using baculovirus expression vectors)yeast cells or mammalian cells. Suitable host cells are discussedfurther in Goeddel (1990) supra. Alternatively, the recombinantexpression vector can be transcribed and translated in vitro, forexample using T7 promoter regulatory sequences and T7 polymerase.

[0124] Expression of proteins in prokaryotes is most often carried outin E. coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein.

[0125] Purified fusion proteins can be utilized in PGC-1 activity assays(e.g., direct assays or competitive assays described in detail below),or to generate antibodies specific for PGC-1 proteins, for example. In apreferred embodiment, a PGC-1 fusion protein expressed in a retroviralexpression vector of the present invention can be utilized to infectbone marrow cells, which are subsequently transplanted into irradiatedrecipients. The pathology of the subject recipient is, then examinedafter sufficient time has passed (e.g., six (6) weeks).

[0126] Examples of suitable inducible non-fusion E. coli expressionvectors include pTrc (Amann et al. (1988) Gene 69:301-315) and pETI 11d(Studier et al. (1990) Methods Enzymol. 185:60-89). Target geneexpression from the pTrc vector relies on host RNA polymerasetranscription from a hybrid trp-lac fusion promoter. Target geneexpression from the pET 11d vector relies on transcription from a T7gn10-lac fusion promoter mediated by a coexpressed viral RNA polymerase(T7 gn1). This viral polymerase is supplied by host strains BL21 (DE3)or HMS174(DE3) from a resident prophage harboring a T7 gn1 gene underthe transcriptional control of the lacUV 5 promoter.

[0127] One strategy to maximize recombinant protein expression in E.coli is to express the protein in a host bacteria with an impairedcapacity to proteolytically cleave the recombinant protein (Gottesman,S. (1990) Methods Enzymol. 185:119-128). Another strategy is to alterthe nucleic acid sequence of the nucleic acid to be inserted into anexpression vector so that the individual codons for each amino acid arethose preferentially utilized in E. coli (Wada et al. (1992) NucleicAcids Res. 20:2111-2118). Such alteration of nucleic acid sequences ofthe invention can be carried out by standard DNA synthesis techniques.

[0128] In another embodiment, the PGC-1 expression vector is a yeastexpression vector. Examples of vectors for expression in yeast S.cerevisiae include pYepSec1 (Baldari et al. (1987) EMBO J. 6:229-234),pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz etal. (1987) Gene 54:113-123), pYES2 (Invitrogen Corp., San Diego,Calif.), and picZ (Invitrogen Corp., San Diego, Calif.).

[0129] Alternatively, PGC-1 proteins can be expressed in insect cellsusing baculovirus expression vectors. Baculovirus vectors available forexpression of proteins in cultured insect cells (e.g., Sf9 cells)include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165)and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).

[0130] In yet another embodiment, a nucleic acid of the invention isexpressed in mammalian cells using a mammalian expression vector.Examples of mammalian expression vector's include pCDM8 (Seed, B. (1987)Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195).When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirusand Simian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17. of Sambrook, J.et al. Molecular Cloning: A Laboratory Manual. 2^(nd) ed, Cold SpringHarbor Laboratory, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989.

[0131] In another embodiment, the recombinant mammalian expressionvector is capable of directing expression of the nucleic acidpreferentially in a particular cell type (e.g., tissue-specificregulatory elements are used to express the nucleic acid).Tissue-specific regulatory elements are known in the art. Non-limitingexamples of suitable tissue-specific promoters include the albuminpromoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277),lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol.43:235-275), in particular promoters of T cell receptors (Winoto andBaltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Baneiji et al.(1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748),neuron-specific promoters (e.g., the neurofilament promoter, Byrne andRuddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477),pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).Developmentally-regulated promoters are also encompassed, for examplethe murine hox promoters (Kessel and Gruss (1990) Science 249:374-379)and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev.3:537-546).

[0132] The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to PGC-1 mRNA. Regulatory sequences operativelylinked to a nucleic acid cloned into the antisense orientation can bechosen which direct the continuous expression of the antisense RNAmolecule in a variety of cell types, for instance viral promoters and/orenhancers, or regulatory sequences can be chosen which directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see Weintraub, H. etal. “Antisense RNA as a molecular tool for genetic analysis”,Reviews—Trends in Genetics, Vol. 1(1) 1986.

[0133] Another aspect of the invention pertains to host cells into whicha PGC-1 nucleic acid molecule of the invention is introduced, e.g., aPGC-1 nucleic acid molecule within a vector (e.g., a recombinantexpression vector) or a PGC-1 nucleic acid molecule containing sequenceswhich allow it to homologously recombine into a specific site of thehost cell's genome. The terms “host cell” and “recombinant host cell”are used interchangeably herein. It is understood that such terms refernot only to the particular subject cell but to the progeny or potentialprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein.

[0134] A host cell can be any prokaryotic or eukaryotic cell. Forexample, a PGC-1 protein can be expressed in bacterial cells such as E.coli, insect cells, yeast or mammalian cells (such as Chinese hamsterovary cells (CHO), COS cells, or C2C12 cells). Other suitable host cellsare known to those skilled in the art.

[0135] Vector DNA can be introduced into prokaryotic or eukaryotic cellsvia conventional transformation or transfection techniques. As usedherein, the terms “transformation” and “transfection” are intended torefer to a variety of art-recognized techniques for introducing foreignnucleic acid (e.g., DNA) into a host cell, including calcium phosphateor calcium chloride co-precipitation, DEAE-dextran-mediatedtransfection, lipofection, or electroporation. Suitable methods fortransforming or transecting host cells can be found in Sambrook et al.(Molecular Cloning: A Laboratory Manual. 2^(nd) ed, Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989), and other laboratory manuals.

[0136] For stable transfection of mammalian cells, it is known that,depending upon the expression vector and transfection technique used,only a small fraction of cells may integrate the foreign DNA into theirgenome. In order to identify and select these integrants, a gene thatencodes a selectable marker (e.g., resistance to antibiotics) isgenerally introduced into the host cells along with the gene ofinterest. Preferred selectable markers include those which conferresistance to drugs, such as G418, hygromycin and methotrexate. Nucleicacid encoding a selectable marker can be introduced into a host cell onthe same vector as that encoding a PGC-1 protein or can be introduced ona separate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

[0137] A host cell of the invention, such as a prokaryotic or eukaryotichost cell in culture, can be used to produce (i.e., express) a PGC-1protein. Accordingly, the invention further provides methods forproducing a PGC-1 protein using the host cells of the invention. In oneembodiment, the method comprises culturing the host cell of theinvention (into which a recombinant expression vector encoding a PGC-1protein has been introduced) in a suitable medium such that a PGC-1protein is produced. In another embodiment, the method further comprisesisolating a PGC-1 protein from the medium or the host cell.

[0138] The host cells of the invention can also be used to producenon-human transgenic animals. For example, in one embodiment, a hostcell of the invention is a fertilized oocyte or an embryonic stem cellinto which PGC-1-coding sequences have been introduced. Such host cellscan then be used to create non-human transgenic animals in whichexogenous PGC-1 sequences have been introduced into their genome orhomologous recombinant animals in which endogenous PGC-1 sequences havebeen altered. Such animals are useful for studying the function and/oractivity of a PGC-1 protein and for identifying and/or evaluatingmodulators of PGC-1 activity. As used herein, a “transgenic animal” is anon-human animal, preferably a mammal, more preferably a rodent such asa rat or mouse, in which one or more of the cells of the animal includesa tangerine. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, amphibians, and the like.A transgene is exogenous DNA which is integrated into the genome of acell from which a transgenic animal develops and which remains in thegenome of the mature animal, thereby directing the expression of anencoded gene product in one or more cell types or tissues of thetransgenic animal. As used herein, a “homologous recombinant animal” isa non-human animal, preferably a mammal, more preferably a mouse, inwhich an endogenous PGC-1 gene has been altered by homologousrecombination between the endogenous gene and an exogenous DNA moleculeintroduced into a cell of the animal, e.g., an embryonic cell of theanimal, prior to development of the animal.

[0139] A transgenic animal of the invention can be created byintroducing a PGC-1-encoding nucleic acid into the male pronuclei of afertilized oocyte, e.g., by microinjection or retroviral infection, andallowing the oocyte to develop in a pseudopregnant female foster animal.The PGC-1 cDNA sequence of SEQ ID NO:6, 8, 12, or 14 can be introducedas a transgene into the genome of a non-human animal. Alternatively, anon-human homologue of a human PGC-1 gene, such as a rat or mouse PGC-1gene, can be used as a transgene. Alternatively, a PGC-1 gene homologue,such as another PGC-1 family member, can be isolated based onhybridization to the PGC-1 cDNA sequences of SEQ ID NO:6, 8, 9, 11, 12,14, 15, or 17, or the DNA insert of the plasmid deposited with ATCC asAccession Number ______ or ______ (described further in subsection Iabove) and used as a transgene. Intronic sequences and polyadenylationsignals can also be included in the transgene to increase the efficiencyof expression of the transgene. A tissue-specific regulatory sequence(s)can be operably linked to a PGC-1 transgene to direct expression of aPGC-1 protein to particular cells. Methods for generating transgenicanimals via embryo manipulation and microinjection, particularly animalssuch as mice, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder etal., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B.,Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1986). Similar methods are used for production ofother transgenic animals. A transgenic founder animal can be identifiedbased upon the presence of a PGC-1 transgene in its genome and/orexpression of PGC-1 mRNA in tissues or cells of the animals. Atransgenic founder animal can then be used to breed additional animalscarrying the transgene. Moreover, transgenic animals carrying atransgene encoding a PGC-1 protein can further be bred to othertransgenic animals carrying other transgenes.

[0140] To create a homologous recombinant animal, a vector is preparedwhich contains at least a portion of a PGC-1 gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the PGC-1 gene. The PGC-1 gene can be a human gene(e.g., the cDNA of SEQ ID NO:6, 8, 12, or 14), but more preferably, is anon-human homologue of a human PGC-1 gene (e.g., a cDNA isolated bystringent hybridization with the nucleotide sequence of SEQ ID NO:6, 8,12, or 14), For example, a mouse PGC-1 gene can be used to construct ahomologous recombination nucleic acid molecule, e.g., a vector, suitablefor altering an endogenous PGC-1 gene in the mouse genome. In apreferred embodiment, the homologous recombination nucleic acid moleculeis designed such that, upon homologous recombination, the endogenousPGC-1 gene is functionally disrupted (i.e., no longer encodes afunctional protein; also referred to as a “knock out” vector).Alternatively, the homologous recombination nucleic acid molecule can bedesigned such that, upon homologous recombination, the endogenous PGC-1gene is mutated or otherwise altered but still encodes functionalprotein (e.g., the upstream regulatory region can be altered to therebyalter the expression of the endogenous PGC-1 protein). In the homologousrecombination nucleic acid molecule, the altered portion of the PGC-1gene is flanked at its 5′ and 3′ ends by additional nucleic acidsequence of the PGC-1 gene to allow for homologous recombination tooccur between the exogenous PGC-1 gene carried by the homologousrecombination nucleic acid molecule and an endogenous PGC-1 gene in acell, e.g., an embryonic stem cell. The additional flanking PGC-1nucleic acid sequence is of sufficient length for successful homologousrecombination with the endogenous gene. Typically, several kilobases offlanking DNA (both at the 5′ and 3′ ends) are included in the homologousrecombination nucleic acid molecule (see, e.g., Thomas, K. R andCapecchi, M. R. (1987) Cell 51:503 for a description of homologousrecombination vectors). The homologous recombination nucleic acidmolecule is introduced into a cell, e.g., an embryonic stem cell line(e.g., by electroporation) and cells in which the introduced PGC-1 genehas homologously recombined with the endogenous PGC-1 gene are selected(see e.g., Li, E. et al. (1992) Cell 69:915). The selected cells canthen be injected into a blastocyst of an animal (e.g., a mouse) to formaggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, Robertson, E. J. ed. (IRL,Oxford, 1987)pp. 113-152). A chimeric embryo can then be implanted intoa suitable pseudopregnant female foster animal and the embryo brought toterm. Progeny harboring the homologously recombined DNA in their germcells can be used to breed animals in which all cells of the animalcontain the homologously recombined. DNA by germline transmission of thetransgene. Methods for constructing homologous recombination nucleicacid molecules, e.g., vectors, or homologous recombinant animals aredescribed further in Bradley, A. (1991) Current Opin. Biotechnol.2:823-829 and in PCT International Publication Nos.: WO 90/11354 by LeMouellec et al.; WO 91/01140 by Smithies et al.; WO 92/0968 by Zijlstraet al.; and WO 93/04169 by Berns et al.

[0141] In another embodiment, transgenic non-humans animals can beproduced which contain selected systems which allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. (1992) Proc.Natl. Acad. Sci. USA 89:6232-6236. Another example of a recombinasesystem is the FLP recombinase system of Saccharomyces cerevisiae(O'Gorman et al. (1991) Science 251:1351-1355). If a cre/loxPrecombinase system is used to regulate expression of the transgene,animals containing transgenes encoding both the Cre recombinase and aselected protein are required. Such animals can be provided through theconstruction of “double” transgenic animals, e.g., by mating twotransgenic animals, one containing a transgene encoding a selectedprotein and the other containing a transgene encoding a recombinase.

[0142] Clones of the non-human transgenic animals described herein canalso be produced according to the methods described in Wilmut, I. et al.(1997) Nature 385:810-813and PCT International Publication Nos. WO97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, fromthe transgenic animal can be isolated and induced to exit the growthcycle and enter G_(O) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyte and then transferred to pseudopregnant femalefoster animal. The offspring borne of this female foster animal will bea clone of the animal from which the cell, e.g. the somatic cell, isisolated.

[0143] IV. Pharmaceutical Compositions

[0144] The PGC-1 nucleic acid molecules, of PGC-1 proteins, fragmentsthereof, anti-PGC-1 antibodies, and PGC-1 modulators (also referred toherein as “active compounds”) of the invention can be incorporated intopharmaceutical compositions suitable for administration. Suchcompositions typically comprise the nucleic acid molecule, protein, orantibody and a pharmaceutically acceptable carrier. As used herein thelanguage “pharmaceutically acceptable carrier” is intended to includeany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsis contemplated. Supplementary active compounds can also be incorporatedinto the compositions.

[0145] A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols,.glycerine,propyleneglycol or other synthetic solvents; antibacterial agents suchas benzyl alcohol or methyl parabens; antioxidants such as ascorbic acidor sodium bisulfite; chelating agents such as ethylenediaminetetraaceticacid; buffers such as acetates, citrates or phosphates and agents forthe adjustment of tonicity such as sodium chloride or dextrose. pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

[0146] Pharmaceutical compositions suitable for injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example; glycerol, propylene glycol, andliquid, polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example; by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

[0147] Sterile injectable solutions can be prepared by incorporating theactive compound (e.g., a fragment of a PGC-1 protein or an anti-PGC-1antibody) in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

[0148] Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or cornstarch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

[0149] For administration by inhalation, the compounds are delivered inthe form of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

[0150] Systemic administration can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

[0151] The compounds can also be prepared in the form of suppositories(e.g., with conventional suppository bases such as cocoa butter andother glycerides) or retention enemas for rectal delivery.

[0152] In one embodiment, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand micro encapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

[0153] It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms, of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

[0154] Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD50 (the dose lethal to50% ofthe population) and the ED50 (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD50/ED50. Compounds which exhibit large therapeutic indices arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

[0155] The data obtained from the cell culture assays and animal studiescan be used in formulating a range of dosage for use in humans. Thedosage of such compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

[0156] As defined herein, a therapeutically effective amount of proteinor polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight. The skilled artisan will appreciate that certainfactors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of a protein, polypeptide, orantibody can include a single treatment or, preferably, can include aseries of treatments.

[0157] In a preferred example, a subject is treated with antibody,protein, or polypeptide in the range of between about 0.1 to 20 mg/kgbody weight, one time per week for between about 1 to 10 weeks,preferably between 2 to 8 weeks, more preferably between about 3 to 7weeks, and even more preferably for about 4, 5, or 6 weeks. It will alsobe appreciated that the effective dosage of antibody, protein, orpolypeptide used for treatment may increase or decrease over the courseof a particular treatment. Changes in dosage may result and becomeapparent from the results of diagnostic assays as described herein.

[0158] The present invention encompasses agents which modulateexpression or activity. An agent may, for example, be a small molecule.For example, such small molecules include, but are not limited to,peptides, peptidomimetics, amino acids, amino acid analogs,polynucleotides, polynucleotide analogs, nucleotides, nucleotideanalogs, organic or inorganic compounds (i.e., including heteroorganicand organometallic compounds) having a molecular weight less than about10,000 grams per mole, organic or inorganic compounds having a molecularweight less than about 5,000 grams per mole, organic or inorganiccompounds having a molecular weight less than about 1,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 500 grams per mole, and salts, esters, and other pharmaceuticallyacceptable forms of such compounds. It is understood that appropriatedoses of small molecule agents depends upon a number of factors withinthe ken of the ordinarily skilled physician, veterinarian, orresearcher. The dose(s) of the small molecule will vary, for example,depending upon the identity, size, and condition of the subject orsample being treated, further depending upon the route by which thecomposition is to be administered, if applicable, and the effect whichthe practitioner desires the small molecule to have upon the nucleicacid or polypeptide of the invention.

[0159] Exemplary doses include milligram or microgram amounts of thesmall molecule per kilogram of subject or sample weight (e.g., about 1microgram per kilogram to about 500 milligrams per kilogram, about 100micrograms per kilogram to about 5 milligrams per kilogram, or about 1microgram per kilogram to about 50 micrograms per kilogram. It isfurthermore understood that appropriate doses of a small molecule dependupon the potency of the small molecule with respect to the expression oractivity to be modulated. Such appropriate doses may be determined usingthe assays described herein. When one or more of these small moleculesis to be administered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian, or researcher may, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

[0160] In certain embodiments of the invention, a modulator of PGC-1activity is administered in combination with other agents (e.g., a smallmolecule), or in conjunction with another, complementary treatmentregime. For example, in one embodiment, a modulator of PGC-1 activity isused to treat PGC-1 associated disorder. Accordingly, modulation ofPGC-1 activity may be used in conjunction with, for example, anotheragent used to treat the disorder.

[0161] Further, an antibody (or fragment thereof) may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent or aradioactive metal ion. A cytotoxin or cytotoxic agent includes any agentthat is detrimental to cells. Examples include taxol, cytochalasin B,germicide D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, midiramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomrannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunonibicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

[0162] The conjugates of the invention can be used for modifying a givenbiological response, the drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such astumor necrosis factor, alpha-interferon, beta-interferon, nerve growthfactor, platelet derived growth factor, tissue plasminogen activator;or, biological response modifiers such as, for example, lymphokines,interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”),granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocytecolony stimulating factor (“G-CSF”), or other growth factors.

[0163] Techniques for conjugating such therapeutic moiety to antibodiesare well known, see, e.g., Amon et al. “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy” in Monoclonal Antibodies AndCancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R Liss, Inc.1985); Hellstrom et al. “Antibodies For Drug Delivery” in ControlledDrug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (MarcelDekker, Inc. 1987); Thorpe “Antibody Carriers Of Cytotoxic Agents InCancer Therapy: A Review” in Monoclonal Antibodies '84: Biological AndClinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985);“Analysis, Results, And Future Prospective Of The Therapeutic Use OfRadiolabeled Antibody In Cancer Therapy” in Monoclonal Antibodies ForCancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16(Academic Press 1985); and Thorpe et al. “The Preparation And CytotoxicProperties Of Antibody-Toxin Conjugates” Immunol. Rev. 62:119-58 (1982).Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980.

[0164] The nucleic acid molecules of the invention can be inserted intovectors and used as gene therapy vectors. Gene therapy vectors can bedelivered to a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

[0165] The pharmaceutical compositions can be included in a container,pack, or dispenser together with instructions for administration.

[0166] V. Uses and Methods of the Invention

[0167] The nucleic acid molecules, proteins, protein homologues, proteinfragments, antibodies, peptides, peptidomimetics, and small moleculesdescribed herein can be used in one or more of the following methods: a)screening assays; b) predictive medicine (e.g., diagnostic assays,prognostic assays, monitoring clinical trials, and pharmacogenetics);and c) methods of treatment (e.g.,therapeutic and prophylactic). Asdescribed herein, a PGC-1 protein of the invention has one or more ofthe following activities (i) interaction with a PGC-1 target molecule;(ii) modulation of intracellular signaling; (iii) modulation of cellularmetabolism; (iv) localization to peroxisomes; (v) modulation of theexpression of genes involved in fatty acid uptake and/or oxidation(e.g., LPL, FAT/CD36, VLACS, AOX, MCAD, and/or MCD); (vi) modulation offatty acid uptake and/or oxidation; (vii) modulation of energyhomeostasis; and/or (viii) modulation of lipid homeostasis.

[0168] The isolated nucleic acid molecules of the invention can be used,for example, to express PGC-1 protein (e.g., via a recombinantexpression vector in a host cell in gene therapy applications), todetect PGC-1 mRNA (e.g., in a biological sample) or a genetic alterationin a PGC-1 gene, and to modulate PGC-1 activity, as described furtherbelow. The PGC-1 proteins can be used to treat disorders characterizedby insufficient or excessive production or transport of a PGC-1 targetmolecule or production of PGC-1 inhibitors, for example, PGC-1associated disorders.

[0169] As used herein, a “PGC-1 associated disorder” includes adisorder, disease or condition which is caused or characterized by amisregulation (e.g., downregulation or upregulation) of PGC-1 activity.PGC-1 associated disorders include disorders, diseases, or conditionsrelated to misregulation (e.g., downregulation or upregulation) of fattyacid uptake and/or oxidation, for example, triglyceride storage disease,steatohepatitis, liver tumors, fatty liver, liver disease, myopathy,hyperlipidemia, hyperlipidemia, dyslipidemia, hypercholesterolemia,hypercholesterolemia, hypertension, stroke, hypertryglyceridemia,hypotriglyceridemia, hyperlipoproteinemia, hypolipoproteinemia, NiemannPick disease, atherosclerosis, cardiovascular disease, coronary arterydisease, obesity, overweight, anorexia, cachexia, diabetes, insulinresistance, hypertension, stroke, pancreatitis, diffuse idiopathicskeletal hyperostosis (DISH), atherogenic lipoprotein phenotype (ALP),epilepsy, and polycystic ovarian syndrome, as well as any disorders,diseases, or conditions which are secondary to those described above.

[0170] PGC-1 associated or related disorders also include disordersaffecting tissues (e.g., heart or muscle) in which PGC-1 protein isexpressed.

[0171] In addition, the PGC-1 proteins can be used to screen fornaturally occurring PGC-1 target molecules, to screen for drugs orcompounds which modulate PGC-1 activity, as well as to treat disorderscharacterized by insufficient or excessive production of PGC-1 proteinor production of PGC-1 protein forms which have decreased, aberrant orunwanted activity compared to PGC-1 wild type protein (e.g., aPGC-1-associated disorder).

[0172] Moreover, the anti-PGC-1 antibodies of the invention can be usedto detect and isolate PGC-1 proteins, regulate the bioavailability ofPGC-1 proteins, and modulate PGC-1 activity.

[0173] A. Screening Assays:

[0174] The invention provides a method (also referred to herein as a“screening assay”) for identifying modulators, i.e., candidate or testcompounds or agents (e.g., peptides, peptidomimetics, small molecules orother drugs)which bind to PGC-1 proteins, have a stimulatory orinhibitory effect on, for example, PGC-1 expression or PGC-1 activity,or have a stimulatory or inhibitory effect on, for example, theexpression or activity of a PGC-1 target molecule.

[0175] In one embodiment, the invention provides assays for screeningcandidate or test compounds which are target molecules of a PGC-1protein or polypeptide or biologically active portion thereof In anotherembodiment, the invention provides assays for screening candidate ortest compounds which bind to or modulate the activity of a PGC-1 proteinor polypeptide or biologically active portion thereof The test compoundsof the present invention can be obtained using any of the numerousapproaches in combinatorial library methods known in the art, including:biological libraries; spatially addressable parallel solid phase orsolution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds (Lam, K. S. (1997) Anticancer Drug Des.12:45).

[0176] Examples of methods for the synthesis of molecular libraries canbe found in the art, for example, in: DeWitt et al. (1993) Proc. Natl.Acad. Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994) J. Med Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (I994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed Engl. 33:2061;and Gallop et al. (1994) J. Med Chem. 37:1233.

[0177] Libraries of compounds may be presented in solution (e.g.,Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria(Ladner U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409),plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) oron phage (Scott and Smith (1990) Science 249:386390); (Devlin (1990)Science 249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA87:6378-382); (Felici (1991) J. Mol. Biol 222:301-310); (Ladner supra).

[0178] In one embodiment, an assay is a cell-based assay in which a cellwhich expresses a PGC-1 protein or biologically active portion thereofis contacted with a test compound and the ability of the test compoundto modulate PGC-1 activity is determined. Determining the ability of thetest compound to modulate PGC-1-activity can be accomplished bymonitoring, for example, expression of genes involved in fatty aciduptake and/or oxidation (e.g., LPL, FAT/CD36, VLACS, AOX, MCAD, and/orMCD), in a cell which expresses PGC-1. The cell, for example, can be ofmammalian origin, e.g., a muscle cell such as a primary muscle cell or aC2C12 myoblast or myotube, or a heart cell such as a cardiomyocyte.

[0179] The ability of the test compound to modulate PGC-1 binding to atarget molecule or to bind to PGC-1 can also be determined. Determiningthe ability of the test compound to modulate PGC-1 binding to a targetmolecule can be accomplished, for example, by coupling the PGC-1 targetmolecule with a radioisotope or enzymatic label such that binding of thePGC-1 target molecule to PGC-1 can be determined by detecting thelabeled PGC-1 target molecule in a complex. Alternatively, PGC-1 couldbe coupled with a radioisotope or enzymatic label to monitor the abilityof a test compound to modulate PGC-1 binding to a PGC-1 target moleculein a complex. Determining the ability of the test compound to bind PGC-1can be accomplished, for example, by coupling the compound with aradioisotope or enzymatic label such that binding of the compound toPGC-1 can be determined by detecting the labeled PGC-1 compound in acomplex. For example, compounds (e.g., PGC-1 target molecules) can belabeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, andthe radioisotope detected by direct counting of radioemission or byscintillation counting. Alternatively, compounds can be enzymaticallylabeled with, for example, horseradish peroxidase, alkaline phosphatase,or luciferase, and the enzymatic label detected by determination ofconversion of an appropriate substrate to product.

[0180] It is also within the scope of this invention to determine theability of a compound (e.g., a PGC-1 target molecule) to interact withPGC-1 without the labeling of any of the interactants. For example, amicrophysiometer can be used to detect the interaction of a compoundwith PGC-1 without the labeling of either the compound or the PGC-1.McConnell, H. M. et al. (1992) Science 257:1906-1912. As used herein, a“microphysiometer” (e.g., Cytosensor) is an analytical instrument thatmeasures the rate at which a cell acidifies its environment using alight-addressable potentiometer sensor (LAPS). Changes in thisacidification rate can be used as an indicator of the interactionbetween a compound and PGC-1.

[0181] In another embodiment, an assay is a cell-based assay comprisingcontacting a cell expressing a PGC-1 target molecule (e.g., a PGC-1polypeptide or a non-PGC-1 potassium channel subunit) with a testcompound and determining the ability of the test compound to modulate(e.g., stimulate or inhibit) the activity of the PGC-1 target molecule.Determining the ability of the test compound to modulate the activity ofa PGC-1 target molecule can be accomplished, for example, by determiningthe ability of a PGC-1 protein to bind to or interact with the PGC-1target molecule, or by determining the ability of a PGC-1 protein tomodulate expression of genes involved in fatty acid uptake and/oroxidation (e.g., LPL, FAT/CD36, VLACS, AOY, MCAD, and/or MCD).

[0182] Determining the ability of the PGC-1 protein, or a biologicallyactive fragment thereof, to bind to or interact with a PGC-1 targetmolecule can be accomplished by one of the methods described above fordetermining direct binding. In a preferred embodiment, determining theability of the PGC-1 protein to bind to or interact with a PGC-1 targetmolecule can be accomplished by determining the activity of the targetmolecule. For example, the activity of the target molecule can bedetermined by detecting induction of a cellular response, detectingcatalytic/enzymatic activity of the target molecule upon an appropriatesubstrate, detecting the induction of a reporter gene (comprising atarget-responsive regulatory element operatively linked to a nucleicacid encoding a detectable marker, e.g., luciferase), or detecting atarget-regulated cellular response (i.e., modulation of fatty aciduptake and/or oxidation).

[0183] In yet another embodiment, an assay of the present invention is acell-free assay in which a PGC-1 protein or biologically active portionthereof is contacted with a test compound and the ability of the testcompound to bind to the PGC-1 protein or biologically active portionthereof is determined. Preferred biologically active portions of thePGC-1 proteins to be used in assays of the present invention includefragments which participate in interactions with non-PGC-1 molecules,e.g., fragments with high surface probability scores. Further preferredPGC-1. proteins include amino acid sequences which are unique to PGC-1bor PGC-1c, e.g., SEQ ID NO:10or SEQ ID NO:16, or fragments thereof.Binding of the test compound to the PGC-1 protein can be determinedeither directly or indirectly as described above. In a preferredembodiment, the assay includes contacting the PGC-1 protein orbiologically active portion thereof with a known compound which bindsPGC-1 to form an assay mixture, contacting the assay mixture with a testcompound, and determining the ability of the test compound to interactwith a PGC-1 protein, wherein determining the ability of the testcompound to interact with a PGC-1 protein comprises determining theability of the test compound to referentially bind to PGC-1 orbiologically active portion thereof as compared to the known compound.

[0184] In another embodiment, the assay is a cell-free assay in which aPGC-1 protein or biologically active portion thereof is contacted with atest compound and the ability of the test compound to modulate (e.g.,stimulate or inhibit) the activity of the PGC-1 protein or biologicallyactive portion thereof is determined. Determining the ability of thetest compound to modulate the activity of a PGC-1 protein can beaccomplished, for example, by determining the ability of the PGC-1protein to bind to a PGC-1 target molecule by one of the methodsdescribed above for determining direct binding. Determining the abilityof the PGC-1 protein to bind to a PGC-1 target molecule can also beaccomplished using a technology such as real-time BiomolecularInteraction Analysis (BIA). Sjolander, S. and Urbaniczky, C. (1991)Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct.Biol. 5:699-705. As used herein, “BIA” is a technology for studyingbiospecific interactions in real time, without labeling any of theinteractants (e.g., BIAcore). Changes in the optical phenomenon ofsurface plasmon resonance (SPR) can be used as an indication ofreal-time reactions between biological molecules.

[0185] In an alternative embodiment, determining the ability of the testcompound to modulate the activity of a PGC-1 protein can be accomplishedby determining the ability of the PGC-1 protein to further modulate theactivity of a downstaeam effector of a PGC-1 target molecule. Forexample, the activity of the effector molecule on an appropriate targetcan be determined or the binding of the effector to an appropriatetarget can be determined as previously described.

[0186] In yet another embodiment, the cell-free assay involvescontacting a PGC-1 protein or biologically active portion thereof with aknown compound which binds the PGC-1 protein to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with the PGC-1 protein, whereindetermining the ability of the test compound to interact with the PGC-1protein comprises determining the ability of the PGC-1 protein topreferentially bind to or modulate the activity of a PGC-1 targetmolecule.

[0187] In more than one embodiment of the above assay methods of thepresent invention, it may be desirable to immobilize either PGC-1 or itstarget molecule to facilitate separation of complexed from uncomplexedforms of one or both of the proteins, as well as to accommodateautomation of the assay. Binding of a test compound to a PGC-1 protein,or interaction of a PGC-1 protein with a target molecule in the presenceand absence of a candidate compound, can be accomplished in any vesselsuitable for containing the reactants. Examples of such vessels includemicrotiter plates, test tubes, and micro-centrifuge tubes. In oneembodiment, a fusion protein can be provided which adds a domain thatallows one or both of the proteins to be bound to a matrix. For example,glutathione-S-transferase/PGC-1 fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized micrometer plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or PGC-1 protein, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotiter plate wells are washed to remove any unbound components, thematrix immobilized in the case, of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level of PGC-1binding or activity determined using standard techniques.

[0188] Other techniques for immobilizing proteins on matrices can alsobe used in the screening assays of the invention. For example, either aPGC-1 protein or a PGC-1 target molecule can be immobilized utilizingconjugation of biotin and streptavidin. Biotinylated PGC-1 protein ortarget molecules can be prepared from biotin-NHS (N-hydroxy-succinimide)using techniques known in the art (e.g., biotinylation kit, PierceChemicals, Rockford, Ill.), and immobilized in the wells ofstreptavidin-coated 96 well plates (Pierce Chemical). Alternatively,antibodies reactive with PGC-1 protein or target molecules but which donot interfere with binding of the PGC-1 protein to its target moleculecan be derivatized to the wells of the plate, and unbound target orPGC-1 protein trapped in the wells by antibody conjugation. Methods fordetecting such complexes, in addition to those described above for theGST-immobilized complexes, include immunodetection of complexes usingantibodies reactive with the PGC-1 protein or target molecule, as wellas enzyme-linked assays which rely on detecting an enzymatic activityassociated with the PGC-1 protein or target molecule.

[0189] In another embodiment, modulators of PGC-1 expression areidentified in a method wherein a cell is contacted with a candidatecompound and the expression of PGC-1 mRNA or protein in the cell isdetermined. The level of expression of PGC-1 mRNA or protein in thepresence of the candidate compound is compared to the level ofexpression of PGC-1 mRNA or protein in the absence of the candidatecompound. The candidate compound can then be identified as a modulatorof PGC-1 expression based on this comparison. For example, whenexpression of PGC-1 mRNA or protein is greater (statisticallysignificantly greater) in the presence of the candidate compound than inits absence, the candidate compound is identified as a stimulator ofPGC-1 mRNA or protein expression. Alternatively, when expression ofPGC-1 mRNA or protein is less (statistically significantly less) in thepresence of the candidate compound than in its absence, the candidatecompound is identified as an inhibitor of PGC-1 mRNA or proteinexpression. The level of PGC-1 mRNA or protein expression in the cellscan be determined by methods described herein for detecting PGC-1 mRNAor protein.

[0190] In yet another aspect of the invention, the PGC-1 proteins can beused as “bait proteins” in a two-hybrid assay or three-hybrid assay(see, e.g, U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993). Oncogene8:1693-1696; and Brent WO 94/10300) to identify other proteins whichbind to or interact with PGC-1 (“PGC-1-binding proteins” or “PGC-1-bp”)and are involved in PGC-1 activity. Such PGC-1-binding proteins are alsolikely to be involved in the propagation of signals by the PGC-1proteins or PGC-1 targets as, for example, downstream elements of aPGC-1-mediated signaling pathway. Alternatively, such PGC-1-bindingproteins may be PGC-1 inhibitors.

[0191] The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different-DNAconstructs. In one construct, the gene that codes for a PGC-1 protein isfused to a gene encoding the DNA binding domain of a known transcriptionfactor (e.g., GAL-4). In the other construct, a DNA sequence, from alibrary of DNA sequences, that encodes an unidentified protein (“prey”or “sample”) is fused to a gene that codes for the activation domain ofthe known transcription factor. If the “bait” and the “prey” proteinsare able to interact, in vivo, forming a PGC-1-dependent complex, theDNA-binding and activation domains of the transcription factor arebrought into close proximity. This proximity allows transcription of areporter gene (e.g., LacZ) which is operably linked to a transcriptionalregulatory site responsive to the transcription factor. Expression ofthe reporter gene can be detected and cell colonies containing thefunctional transcription factor can be isolated and used to obtain thecloned gene which encodes the protein which interacts with the PGC-1protein.

[0192] In another aspect, the invention pertains to a combination of twoor more of the assays described herein. For example, a modulating agentcan be identified using a cell-based or a cell-free assay, and theability of the agent to modulate the activity of a PGC-1 protein can beconfirmed in vivo, e.g., in an animal such as an animal model for fattyacid metabolism. Such animal models are known in the art and includemice deficient in various genes involved in fatty acid uptake and/oroxidation, for example, mice deficient for peroxisomal Acyl-CoA oxidase(Fan, C. Y. et al. (1998) J. Biol. Chem. 273(25):15639-45) or thetranscription factor PPARα, or mice overexpressing lipoprotein lipase inskeletal muscle and heart.

[0193] This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a PGC-1 modulating agent, an antisense PGC-1nucleic acid molecule, a PGC-1-specific antibody, or a PGC-1 bindingpartner) can be used in an animal model to determine the efficacy,toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal model to determine the mechanism of action of such an agent.Furthermore, this invention pertains to uses of novel agents identifiedby the above-described screening assays for treatments as describedherein.

[0194] B. Detection Assays

[0195] Portions or fragments of the cDNA sequences identified herein(and the corresponding complete gene sequences) can be used in numerousways as polynucleotide reagents. For example, these sequences can beused to: (i) map their respective genes on a chromosome; and, thus,locate gene regions associated with genetic disease; (ii) identify anindividual from a minute biological sample (tissue typing); and (iii)aid in forensic identification of a biological sample. Theseapplications are described in the subsections below.

[0196] 1. Chromosome Mapping

[0197] Once the sequence (or a portion of the sequence) of a gene hasbeen isolated, this sequence can be used to map the location of the geneon a chromosome. This process is called chromosome mapping. Accordingly,portions or fragments of the PGC-1 nucleotide sequences, describedherein, can be used to map the location of the PGC-1 genes on achromosome. The mapping of the PGC-1 sequences to chromosomes is animportant first step in correlating these sequences with genesassociated with disease.

[0198] Briefly, PGC-1 genes can be mapped to chromosomes by preparingPCR primers (preferably 15-25 nucleotides in length) from the PGC-1nucleotide sequences. Computer analysis of the PGC-1 sequences can beused to predict primers that do not span more than one exon in thegenomic DNA, thus complicating the amplification process. These primerscan then be used for PCR screening of somatic cell hybrids containingindividual human chromosomes. Only those hybrids containing the humangene corresponding to the PGC-1 sequences will yield an amplifiedfragment.

[0199] Somatic cell hybrids are prepared by fusing somatic cells fromdifferent mammals (e.g., human and mouse cells). As hybrids of human andmouse cells grow and divide, they gradually lose human chromosomes inrandom order, but retain the mouse chromosomes. By using media in whichmouse cells cannot grow, because they lack a particular enzyme, buthuman cells can, the one human chromosome that contains the geneencoding the needed enzyme, will be retained. By using various media,panels of hybrid cell lines can be established. Each cell line in apanel contains either a single human chromosome or a small number ofhuman chromosomes, and a fill set of mouse chromosomes, allowing easymapping of individual genes to specific human chromosomes (D'EustachioP. et al. (1983) Science 220:919-924). Somatic cell hybrids containingonly fragments of human chromosomes can also be produced by using humanchromosomes with translocations and deletions.

[0200] PCR mapping of somatic cell hybrids is a rapid procedure forassigning a particular sequence to a particular chromosome. Three ormore sequences can be assigned per day using a single thermal cycler.Using the PGC-1 nucleotide sequences to design oligonucleotide primers,sublocalization can be achieved with panels of fragments from specificchromosomes. Other mapping strategies which can similarly be used to mapa PGC-1 sequence to its chromosome include in situ hybridization(described in Fan, Y. et al. (1990) Proc. Natl. Acad. Sci. USA87:6223-27), pre-screening with labeled flow-sorted chromosomes, andpre-selection by hybridization to chromosome-specific cDNA libraries.

[0201] Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location in one step. Chromosome spreads can be made usingcells whose division has been blocked in metaphase by a chemical such ascolcemid that disrupts the mitotic spindle. The chromosomes can betreated briefly with trypsin, and then stained with Giemsa. A pattern oflight and dark bands develops on each chromosomes so that thechromosomes can be identified individually. The FISH technique can beused with a DNA sequence as short as 500 or 600. bases. However, cloneslarger than 1,000 bases have a higher likelihood of binding to a uniquechromosomal location with sufficient signal intensity for simpledetection. Preferably 1000 bases, and more preferably 2,000 bases willsuffice to get good results at a reasonable amount of time. For a reviewof this technique, see Verma et al., Human Chromosomes: A Manual ofBasic Technique (Pergamon Press, New York 1988).

[0202] Reagents for chromosome mapping can be used individually to marka single chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

[0203] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data (such data are found, for example, inMcKusick, V., Mendelian Inheritance in Man, available on-line throughJohns Hopkins University Welch Medical Library). The relationshipbetween a gene and a disease, mapped to the same chromosomal region, canthen be identified through linkage analysis (co-inheritance ofphysically adjacent genes), described in, for example, Egeland, J. etal. (1987) Nature 325:783-787.

[0204] Moreover, differences in the DNA sequences between individualsaffected and unaffected with a disease associated with the PGC-1 gene,can be determined. If a mutation is observed in some or all of theaffected individuals but not in any unaffected individuals, then themutation is likely to be the causative agent of the particular disease.Comparison of affected and unaffected individuals generally involvesfirst looking for structural alterations in the chromosomes, such asdeletions or translocations that are visible from chromosome spreads ordetectable using PCR based on that DNA sequence. Ultimately, completesequencing of genes from several individuals can be performed to confirmthe presence of a mutation and to distinguish mutations frompolymorphisms.

[0205] 2. Tissue Typing

[0206] The PGC-1 sequences of the present invention can also be used toidentify individuals from minute biological samples. The United Statesmilitary, for example, is considering the use of restriction fragmentlength polymorphism (RFLP) for identification of its personnel. In thistechnique, an individual's genomic DNA is digested with one or morerestriction enzymes, and probed on a Southern blot to yield unique bandsfor identification. This method does not suffer from the currentlimitations of “Dog Tags” which can be lost, switched, or stolen, makingpositive identification difficult. The sequences of the presentinvention are useful us additional DNA markers for RFLP (described inU.S. Pat. No. 5,272,057).

[0207] Furthermore, the sequences of the present invention can be usedto provide an alternative technique which determines the actualbase-by-base DNA sequence of selected portions of an individual'sgenome. Thus, the PGC-1. nucleotide sequences described herein can beused to prepare two PCR primers from the 5′ and 3′ ends of thesequences. These primers can then be used to amplify an individual's DNAand subsequently sequence it.

[0208] Panels of corresponding DNA sequences from individuals, preparedin this manner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The sequences of the present invention can be used toobtain such identification sequences from individuals and from tissue.The PGC-1 nucleotide sequences of the invention uniquely representportions of the human genome. Allelic variation occurs to some degree inthe coding regions of these sequences, and to a greater degree in thenoncoding regions. It is estimated that allelic variation betweenindividual humans occurs with a frequency of about once per each 500bases. Each of the sequences described herein can, to some degree, beused as a standard against which DNA from an individual can be comparedfor identification purposes. Because greater numbers of polymorphismsoccur in the noncoding regions, fewer sequences are necessary todifferentiate individuals. The noncoding sequences of SEQ I) NO:6, 9,12, or 15 can comfortably provide positive individual identificationwith a panel of perhaps 10 to 1,000 primers which each yield a noncodingamplified sequence of 100 bases. If predicted coding sequences, such asthose in SEQ ID NO:8, 11, 14, or 17 are used, a more appropriate numberof primers for positive individual identification would be 500-2,000.

[0209] If a panel of reagents from PGC-1 nucleotide sequences describedherein is used to generate a unique identification database for anindividual, those same reagents can later be used to identify tissuefrom that individual. Using the unique identification database, positiveidentification of the individual, living or dead, can be made fromextremely small tissue samples.

[0210] 3. Use of Partial PGC-1 Sequences in Forensic Biology

[0211] DNA-based identification techniques can also be used in forensicbiology. Forensic biology is a scientific field employing genetic typingof biological evidence found at a crime scene as a means for positivelyidentifying, for example, a perpetrator of a crime. To make such anidentification, PCR technology can be used to amplify DNA sequencestaken from very small biological samples such as tissues; e.g., hair orskin, or body fluids, e.g., blood, saliva, or semen found at a crimescene. The amplified sequence can then be compared to a standard,thereby allowing identification of the origin of the biological sample.

[0212] The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e., another DNA sequence that is unique to aparticular individual). As mentioned above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions of SEQ ID NO:1 are particularlyappropriate for this use as greater numbers of polymorphisms occur inthe noncoding regions, making it easier to differentiate individualsusing this technique. Examples of polynucleotide reagents include thePGC-1 nucleotide sequences or portions thereof, e.g., fragments derivedfrom the noncoding regions of SEQ ID NO:1 having a length of at least 20bases, preferably at least 30 bases.

[0213] The PGC-1 nucleotide sequences described herein can further beused to provide polynucleotide reagents, e.g., labeled or labelableprobes which can be used in, for example, an in situ hybridizationtechnique, to identify a specific tissue, e.g. a tissue which expressesPGC-1, such as heart or muscle. This can be very useful in cases where aforensic pathologist is presented with a tissue of unknown origin.Panels of such PGC-1 probes can be used to identify tissue by speciesand/or by organ type.

[0214] In a similar fashion, these reagents, e.g., PGC-1 primers orprobes can be used to screen tissue culture for contamination (i.e.,screen for the presence of a mixture of different types of cells in aculture).

[0215] C. Predictive Medicine:

[0216] The present invention also pertains to the field of predictivemedicine in which diagnostic assays, prognostic assays, and monitoringclinical trials are used for prognostic (predictive) purposes to therebytreat an individual prophylactically. Accordingly, one aspect of thepresent invention relates to diagnostic assays for determining PGC-1protein and/or nucleic acid expression as well as PGC-1 activity, in thecontext of a biological sample (e.g. blood, serum, cells, or tissue) tothereby determine whether an individual is afflicted with a disease ordisorder (e.g., a PGC-1 associated disorder), or is at risk ofdeveloping a disorder, associated with aberrant or unwanted PGC-1expression or activity. The invention also provides for prognostic (orpredictive) assays for determining whether an individual is at risk ofdeveloping a disorder associated with PGC-1 protein, nucleic acidexpression, or activity. For example, mutations in a PGC-1 gene can beassayed in a biological sample. Such assays can be used for prognosticor predictive purpose to thereby prophylactically treat an individualprior to the onset of a disorder characterized by or associated withPGC-1 protein, nucleic acid expression or activity.

[0217] Another aspect of the invention pertains to monitoring theinfluence of agents (e.g., drugs, compounds) on the expression oractivity of PGC-1 in clinical trials.

[0218] These and other agents are described in further detail in thefollowing sections.

[0219] 1. Diagnostic Assays

[0220] An exemplary method for detecting the presence or absence ofPGC-1 protein, polypeptide or nucleic acid in a biological sampleinvolves obtaining a biological sample from a test subject andcontacting the biological sample with a compound or an agent capable ofdetecting PGC-1 protein, polypeptide or nucleic acid (e.g., mRNA,genomic DNA) that encodes PGC-1 protein such that the presence of PGC-1protein or nucleic acid is detected in the biological sample. In anotheraspect, the present invention provides a method for detecting thepresence of PGC-1 activity in a biological sample by contacting thebiological sample with an agent capable of detecting an indicator ofPGC-1 activity such that the presence of PGC-1 activity is detected inthe biological sample. A preferred agent for detecting PGC-1 mRNA orgenomic DNA is a labeled nucleic acid probe capable of hybridizing toPGC-1RNA or genomic DNA. The nucleic acid probe can be, or example, afull-length PGC-1 nucleic acid, such as the nucleic acid of SEQ ID NO:6,8, 9, 11, 12, 14, 15, or 17, or the DNA insert of the plasmid depositedwith ATCC as Accession Number or, or a portion thereof, such as anoligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides inlength and sufficient to specifically hybridize under stringentconditions to PGC-1 mRNA or genomic DNA. Other suitable probes for usein the diagnostic assays of the invention are described herein.

[0221] A preferred agent for detecting PGC-1 protein is an antibodycapable of binding to PGC-1 protein, preferably an antibody with adetectable label. Antibodies can be polyclonal, or more preferably,monoclonal. An intact antibody, or a fragment thereof (e.g., Fab orF(ab′)₂) can be used. The term “labeled”, with regard to the probe orantibody, is intended to encompass direct labeling of the probe orantibody by coupling (i.e., physically linking) a detectable substanceto the probe or antibody, as well as indirect labeling of the probe orantibody by reactivity with another reagent that is directly labeled.Examples of indirect labeling include detection of a primary antibodyusing a fluorescently labeled secondary antibody and end-labeling of aDNA probe with biotin such that it can be detected with fluorescentlylabeled streptavidin. The term “biological sample” is intended toinclude tissues, cells and biological fluids isolated from a subject, aswell as tissues, cells and fluids present within a subject. That is, thedetection method of the invention can be used to detect PGC-1 mRNA,protein, or genomic DNA in a biological sample in vitro as well as invivo. For example, in vitro techniques for detection of PGC-1 mRNAinclude Northern hybridizations and in situ hybridizations. In vitrotechniques for detection of PGC-1 protein include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations andimmunofluorescence. In vitro techniques for detection of PGC-1 genomicDNA include Southern hybridizations. Furthermore, in vivo techniques fordetection of a PGC-1 protein include introducing into a subject alabeled anti-PGC-1 antibody. For example, the antibody can be labeledwith a radioactive marker whose presence and location in a subject canbe detected by standard imaging techniques.

[0222] The present invention also provides diagnostic assays foridentifying the presence or absence of a genetic alterationcharacterized by at least one of (i) aberrant modification or mutationof a gene encoding a PGC-1 protein; (ii) aberrant expression of a geneencoding a PGC-1 protein; (iii) miss-regulation of the gene; and (iii)aberrant post-translational modification of a PGC-1 protein, wherein awild-type form of the gene encodes a protein with a PGC-1 activity.“Misexpression or aberrant expression”, as used herein, refers to anon-wild type pattern of gene expression, at the RNA or protein level.It includes, but is not limited to, expression at non-wild type levels(e.g.,over or under expression); a pattern of expression that differsfrom wild type in terms of the time or stage at which the gene isexpressed (e.g., increased or decreased expression (as compared withwild type) at a predetermined developmental period or stage); a patternof expression that differs from wild type in terms of decreasedexpression (as compared with wild type) in a predetermined cell type ortissue type; a pattern of expression that differs from wild type interms of the splicing size, amino acid sequence, post-transitionalmodification, or biological activity of the expressed polypeptide; apattern of expression that differs from wild type in terms of the effectof an environmental stimulus or extracellular stimulus on expression ofthe gene (e.g., a pattern of increased or decreased expression (ascompared with wild type) in the presence of an increase or decrease inthe strength of the stimulus).

[0223] In one embodiment, the biological sample contains proteinmolecules from the test subject. Alternatively, the biological samplecan contain mRNA molecules from the test subject or genomic DNAmolecules from the test subject A preferred biological sample is a serumsample isolated by conventional means from a subject.

[0224] In another embodiment, the methods further involve obtaining acontrol biological sample from a control subject, contacting the controlsample with a compound or agent capable of detecting PGC-1 protein,mRNA, or genomic DNA, such that the presence of PGC-1 protein, mRNA orgenomic DNA is detected in the biological sample, and comparing thepresence of PGC-1 protein, mRNA or genomic DNA in the control samplewith the presence of PGC-1 protein, mRNA or genomic DNA in the testsample.

[0225] The invention also encompasses kits for detecting the presence ofPGC-1 in a biological sample. For example, the kit can comprise alabeled compound or agent capable of detecting PGC-1 protein or mRNA ina biological sample; means for determining the amount of PGC-1 in thesample; and means for comparing the amount of PGC-1 in the sample with astandard. The compound or agent can be packaged in a suitable container.The kit an further comprise instructions for using the kit to detectPGC-1 protein or nucleic acid.

[0226] 2. Prognostic Assays

[0227] The diagnostic methods described herein can furthermore beutilized to identify subjects having or at risk of developing a diseaseor disorder (e.g., a PGC-1 associated disorder) associated with aberrantor unwanted PGC-1 expression or activity. As used herein, the term“aberrant” includes a PGC-1 expression or activity which deviates fromthe wild type PGC-1 expression or activity. Aberrant expression oractivity includes increased or decreased expression or activity, as wellas expression or activity which does not follow the wild typedevelopmental pattern of expression or the subcellular pattern ofexpression. For example, aberrant PGC-1 expression or activity isintended to include the cases in which a mutation in the PGC-1 genecauses the PGC-1 gene to be under-expressed or over-expressed andsituations in which such mutations result in a non-functional PGC-1protein or a protein which does not function in a wild-type fashion,e.g., a protein which does not interact with a PGC-1 target molecule, orone which interacts with a non-PGC-1 target molecule. As used herein,the term “unwanted” includes an unwanted phenomenon involved in abiological response such as deregulated fatty acid uptake and/oroxidation. For example, the term unwanted includes a PGC-1 expression oractivity which is undesirable in a subject.

[0228] The assays described herein, such as the preceding diagnosticassays or the following assays, can be utilized to identify a subjecthaving or at risk of developing a disorder associated with amisregulation in PGC-1 protein activity or nucleic acid expression, suchas a PGC-1 associated. Alternatively, the prognostic assays can beutilized to identify a subject having or at risk for developing adisorder associated with a misregulation in PGC-1 protein activity ornucleic acid expression, such as a PGC-1 associated. Thus, the presentinvention provides a method for identifying a disease or disorderassociated with aberrant or unwanted PGC-1 expression or activity inwhich a test sample is obtained from a subject and PGC-1 protein ornucleic acid (e.g, mRNA or genomic DNA) is detected, wherein thepresence of PGC-1 protein or nucleic acid is diagnostic for a subjecthaving or at risk of developing a disease or disorder associated withaberrant or unwanted PGC-1 expression or activity. As used herein, a“test sample” refers to a biological sample obtained from a subject ofinterest. For example, a test sample can be a biological fluid (e.g.,serum), cell sample, or tissue.

[0229] Furthermore, the prognostic assays described herein can be usedto determine whether a subject can be administered an agent (e.g.,anagonist, antagonist, peptidomimetic, protein; peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant or unwanted PGC-1 expression or activity. Forexample, such methods can be used to determine whether a subject can beeffectively treated with an agent for a drug or toxin sensitivitydisorder or a cell proliferation and/or differentiation disorder. Thus,the present invention provides methods for determining whether a subjectcan be effectively treated with an agent for a disorder associated withaberrant or unwanted PGC-1 expression or activity in which a test sampleis obtained and PGC-1 protein or nucleic acid expression or activity isdetected (e.g., wherein the abundance of PGC-1 protein or nucleic acidexpression or activity is diagnostic for a subject that can beadministered the agent to treat a disorder associated with aberrant orunwanted PGC-1 expression or activity).

[0230] The methods of the invention can also be used to detect geneticalterations in a PGC-1 gene, thereby determining if a subject with thealtered gene is at risk for a disorder characterized by misregulation inPGC-1 protein activity or nucleic acid expression, such as a PGC-1associated disorder. In preferred embodiments, the methods includedetecting, in a sample of cells from the subject, the presence orabsence of a genetic alteration characterized by at least one of analteration affecting the integrity of a gene encoding a PGC-1-protein,or the mis-expression of the PGC-1 gene. For example, such geneticalterations can be detected by ascertaining the existence of at leastone of 1) a deletion of one or more nucleotides from a PGC-1 gene; 2) anaddition of one or more nucleotides to a PGC-1 gene; 3) a substitutionof one or more nucleotides of a PGC-1 gene, 4) a chromosomalrearrangement of a PGC-1 gene; 5) an alteration in the level of amessenger RNA transcript of a PGC-1 gene, 6) aberrant modification of aPGC-1 gene, such as of the methylation pattern of the genomic DNA, 7)the presence of a non-wild type splicing pattern of a messenger RNAtranscript of a PGC-1 gene, 8) a non-wild type level of a PGC-1-protein,9) allelic loss of a PGC-1 gene, and 10) inappropriatepost-translational modification of a PGC-1-protein As described herein,there are a large number of assays known in the art which can be usedfor detecting alterations in a PGC-1 gene. A preferred biologicalsamplers a tissue or serum sample isolated by conventional means from asubject.

[0231] In certain embodiments, detection of the alteration involves theuse of a probe/primer in a polymerase chain reaction (PCR) (see, e.g.,U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR,or, alternatively, in a ligation chain reaction (LCR) (see, e.g.,Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al.(1994) Proc. Natl. Acad. Sci. USA 91:360-364), the latter of which canbe particularly useful for detecting point mutations in the PGC-1-gene(see Abravaya et al. (1995) Nucleic Acids Res. 23:675-682). This methodcan include the steps of collecting a sample of cells from a subject,isolating nucleic acid (e.g., genomic, mRNA or both) from the cells ofthe sample, contacting the nucleic acid sample with one or more primerswhich specifically hybridize to a PGC-1 gene under conditions such thathybridization and amplification of the PGC-1-gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. It is anticipated that PCR and/or LCR may bedesirable to use as a preliminary amplification step in conjunction withany of the techniques used for detecting mutations described herein.

[0232] Alternative amplification methods include: self sustainedsequence replication (Guatelli, J. C. et al. (1990) Proc. Natl. Acad.Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D.Y. et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-BetaReplicase (Lizardi, P. M. et al. (1988) Bio-Technology 6:1.197), or anyother nucleic acid amplification method, followed by the detection ofthe amplified molecules using techniques well known to those of skill inthe art. These detection schemes are especially useful for the detectionof nucleic acid molecules if such molecules are present in very lownumbers.

[0233] In an alternative embodiment, mutations in a PGC-1 gene from asample cell can be identified by alterations in restriction enzymecleavage patterns. For example, sample and control DNA is isolated,amplified (optionally), digested with one or more restrictionendonucleases, and fragment length sizes are determined by gelelectrophoresis and compared. Differences in fragment length sizesbetween sample and control DNA indicates mutations in the sample DNA.Moreover, the use of sequence specific ribozymes (see, for example, U.S.Pat. No. 5,498,531) can be used to score for the presence of specificmutations by development or loss of a ribozyme cleavage site.

[0234] In other embodiments, genetic mutations in PGC-1 can beidentified by hybridizing a sample and control nucleic acids, e.g. DNAor RNA, to high density arrays containing hundreds or thousands ofoligonucleotides probes (Cronin, M. T. et al. (1996) Hum. Mutat.7:244-255; Kozal, M. J. et al. (1996) Nat. Med 2:753-759). For example,genetic mutations in PGC-1 can be identified in two dimensional arrayscontaining light-generated DNA probes as described in Cronin et al.(1996) supra Briefly, a first hybridization array of probes can be usedto scan through long stretches of DNA in a sample and control toidentify base changes between the sequences by making linear arrays ofsequential overlapping probes. This step allows the identification ofpoint mutations. This step is followed by a second hybridization arraythat allows the characterization of specific mutations by using smaller,specialized probe arrays complementary to all variants or mutationsdetected. Each mutation array is composed of parallel probe sets, onecomplementary to the wild-type gene and the other complementary to themutant gene.

[0235] In yet another embodiment, any of a variety of sequencingreactions known in the art can be used to directly sequence the PGC-1gene and detect mutations by comparing the sequence of the sample PGC-1with the corresponding wild-type (control) sequence. Examples ofsequencing reactions include those based on techniques developed byMaxam and Gilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger((1977) Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplatedthat any of a variety of automated sequencing procedures can be utilizedwhen performing the diagnostic assays (Naeve, C. W. (1995) Biotechniques19:448), including sequencing by mass spectrometry (see, e.g., PCTInternational Publication No. WO 94/16101; Cohen et al. (1996) Adv.Chromatogr. 36:127-162; and Griffin et al. (1993) Appl. Biochem.Biotechnol. 38:147-159).

[0236] Other methods for detecting mutations in the PGC-1 gene includemethods in which protection from cleavage agents is used to detectmismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers. et al.(1985) Science 230:1242). In general, the art technique of “mismatchcleavage” starts by providing heteroduplexes formed by hybridizing(labeled) RNA or DNA containing the wild-type PGC-1 sequence withpotentially mutant RNA or DNA obtained from a tissue sample. Thedouble-stranded duplexes are treated with an agent which cleavessingle-stranded regions of the duplex such as which will exist due tobasepair mismatches between the control and sample strands. Forinstance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybridstreated with S1 nuclease to enzymatically digesting the mismatchedregions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can betreated with hydroxylamine or osmium tetroxide and with piperidine inorder to digest mismatched regions. After digestion of the mismatchedregions, the resulting material is then separated by size on denaturingpolyacrylamide gels to determine the site of mutation. See, for example,Cotton et al. (1988) Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al.(1992) Methods Enzymol. 217:286-295. In a preferred embodiment, thecontrol DNA or RNA can be labeled for detection.

[0237] In still another embodiment, the mismatch cleavage reactionemploys one or more proteins that recognize mismatched base pairs indouble-stranded DNA (so called “DNA mismatch repair” enzymes).in definedsystems for detecting and mapping point mutations in PGC-1 cDNAsobtained from samples of cells. For example, the mutY enzyme of E. colicleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLacells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis15:1657-1662). According to an exemplary embodiment, a probe based on aPGC-1 sequence, e.g., a wild-type PGC-1 sequence, is hybridized to acDNA or other DNA product from a test cell(s). The duplex is treatedwith a DNA mismatch repair enzyme, and the cleavage products, if any,can be detected from electrophoresis protocols or the like. See, forexample, U.S. Pat. No. 5,459,039.

[0238] In other embodiments, alterations in electrophoretic mobilitywill be used to identify mutations in PGC-1 genes. For example, singlestrand conformation polymorphism (SSCP) maybe used to detect differencesin electrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc. Natl. Acad. Sci. USA 86:2766; see also Cotton(1993) Mutat. Res. 285:125-144; and Hayashi (1992) Genet. Anal. TechAppl. 9:73-79). Single-stranded DNA fragments of sample and controlPGC-1 nucleic acids will be denatured and allowed to renature. Thesecondary structure of single-stranded nucleic acids varies according tosequence, the resulting alteration in electrophoretic mobility enablesthe detection of even a single base change The DNA fragments may belabeled or detected with labeled probes. The sensitivity of the assaymay be enhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).

[0239] In yet another embodiment the movement of mutant or wild-typefragments in polyacrylamide gels containing a gradient of denaturant isassayed using denaturing, gradient gel electrophoresis (DGGE) (Myers etal. (1985) Nature 313:495). When DGGE is used as the method of analysis,DNA will be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys. Chem. 265:12753).

[0240] Examples of other techniques for detecting point mutationsinclude, but are not limited to, selective oligonucleotidehybridization, selective amplification, or selective primer extension.For example, oligonucleotide primers may be prepared in which the knownmutation is placed centrally and then hybridized to target DNA underconditions which permit hybridization only if a perfect match is found(Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl.Acad. Sci. USA 86:6230). Such allele specific oligonucleotides arehybridized to PCR amplified target DNA or a number of differentmutations when the oligonucleotides are attached to the hybridizingmembrane and hybridized with labeled target DNA.

[0241] Alternatively, allele specific amplification technology whichdepends on selective PCR amplification may be used in conjunction withthe instant invention. Oligonucleotides used as primers for specificamplification may carry the mutation of interest in the center of themolecule (so that amplification depends on differential hybridization)(Gibbs et al. (1989) Nucleic Acids. Res. 17:2437-2448) or at the extreme3′ end of one primer where, under appropriate conditions, mismatch canprevent, or reduce polymerase extension (Prossner (1993) Tibtech11:238). In addition it may be desirable to introduce a novelrestriction site in the region of the mutation to create cleavage-baseddetection (Gasparini et al. (1992) Mol. Cell Probes 6:1). It isanticipated that in certain embodiments amplification may also beperformed using Taq ligase for amplification (Barany (1991) Proc. Natl.Acad. Sci. USA 88:189). In such cases, ligation will occur only if thereis a perfect match at the 3′ end of the 5′ sequence making it possibleto detect the presence of a known mutation at a specific site by lookingfor the presence or absence of amplification.

[0242] The methods described herein may be performed, for example, byutilizing pre-packaged diagnostic kits comprising at least one probenucleic acid or antibody reagent described herein, which may beconveniently used, e.g., in clinical settings to diagnose patientsexhibiting symptoms or family history of a disease or illness involvinga PGC-1 gene.

[0243] Furthermore, any cell type or tissue in which PGC-1 is expressed(e.g., heart or muscle) may be utilized in the prognostic assaysdescribed herein.

[0244] 3. Monitoring of Effects During Clinical Trials

[0245] Monitoring the influence of agents (e.g., drugs) on theexpression or activity of a PGC-1 protein (e.g., the modulation of PGC-1activity, expression of fatty acid uptake and/or oxidation genes, and/orfatty acid uptake and/or oxidation mechanisms) can be applied not onlyin basic drug screening, but also in clinical trials. For example, theeffectiveness of an agent determined by a screening assay as describedherein to increase PGC-1 gene expression, protein levels, or upregulatePGC-1 activity, can be monitored in clinical trials of subjectsexhibiting decreased PGC-1 gene expression, protein levels, ordownregulated PGC-1 activity. Alternatively, the effectiveness of anagent determined by a screening assay to decrease PGC-1 gene expression,protein levels, or downregulate PGC-1 activity, can be monitored inclinical trials of subjects exhibiting increased PGC-1 gene expression,protein levels, or unregulated PGC-1 activity. In such clinical trials,the expression or activity of a PGC-1 gene, and preferably, other genesthat have been implicated in, for example, a PGC-1-associated disordercan be used as a “read out” or markers of the phenotype of a particularcell.

[0246] For example, and not by way of limitation, genes, includingPGC-1, that are modulated in cells by treatment with an agent (e.g.,compound, drug or small molecule) which modulates PGC-1 activity (e.g.,identified in a screening assay as described herein) can be identified.Thus, to study the effect of agents on PGC-1-associated disorders (e.g.,disorders characterized by deregulated PGC-1 activity, expression offatty acid uptake and/or oxidation genes, and/or fatty acid uptakeand/or oxidation mechanisms), for example, in a clinical trial, cellscan be isolated and RNA prepared and analyzed for the levels ofexpression of PGC-1 and other genes implicatedin the PGC-1-associateddisorder, respectively. The levels of gene expression (e.g., a geneexpression pattern) can be quantified by northern blot analysis orRT-PCR, as described herein, or alternatively by measuring the amount ofprotein produced, by one of the methods as described herein, or bymeasuring the levels of activity of PGC-1 or other genes. In this way,the gene expression pattern can serve as a marker, indicative of thephysiological response of the cells to the agent. Accordingly, thisresponse state may be determined before, and at various points duringtreatment of the individual with the agent.

[0247] In a preferred embodiment, the present invention provides amethod for monitoring the effectiveness of treatment of a subject withan agent (e.g., an agonist, antagonist, peptidomimetic, protein,peptide, nucleic acid, small molecule, or other drug candidateidentified by the screening assays described herein) including the stepsof (i) obtaining a pre-administration sample from a subject prior toadministration of the agent; (ii) detecting the level of expression of aPGC-1 protein, mRNA, or genomic DNA in the preadministration sample;(iii) obtaining one or more post-administration samples from thesubject; (iv) detecting the level of expression or activity of the PGC-1protein, mRNA, or genomic DNA in the post-adminisration samples; (v)comparing the level of expression or activity of the PGC-1 protein,mRNA, or genomic DNA in the pre-administration sample with the PGC-1protein, mRNA, or genomic DNA in the post administration sample orsamples; and (vi) altering the administration of the agent to thesubject accordingly. For example, increased administration of the agentmay be desirable to increase the expression or activity of PGC-1 tohigher levels than detected, i.e., to increase the effectiveness of theagent. Alternatively, decreased administration of the agent may bedesirable to decrease expression or activity of PGC-1 to lower levelsthan detected, i.e., to decrease the effectiveness of the agent.According to such an embodiment, PGC-1 expression or activity may beused as an indicator of the effectiveness of an agent, even in theabsence of an observable phenotypic response.

[0248] D. Methods of Treatment:

[0249] The present invention provides for both prophylactic andtherapeutic methods of treating a subject at risk of (or susceptible to)a disorder or having a PGC-1-associated disorder, e.g., a disorderassociated with aberrant or unwanted PGC-1 expression or activity (e.g.,a PGC-1 associated disorder). As used herein, “treatment” of a subjectincludes the application or administration of a therapeutic agent to asubject, or application or administration of a therapeutic agent to acell or tissue from a subject, who has a diseases or disorder, has asymptom of a disease or disorder, or is at risk of (or susceptible to) adisease or disorder, with the purpose to cure, heal, alleviate, relieve,alter, remedy, ameliorate, improve, or affect the disease or disorder,the symptom of the disease or disorder, or the risk of (orsusceptibility to) the disease or disorder. As used herein, a“therapeutic agent” includes, but is not limited to, small molecules,peptides, polypeptides, antibodies, ribozymes, and antisenseoligonucleotides.

[0250] With regards to both prophylactic and therapeutic methods oftreatment, such treatments may be specifically tailored or modified,based on knowledge obtained from the field of pharmacogenomics.“Pharmacogenomics”, as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers the study of how a patient'sgenes determine his or her response to a drug (e.g., a patient's “drugresponse phenotype”, or “drug response genotype”). Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with either the PGC-1 molecules ofthe present invention or PGC-1 modulators according to that individual'sdrug response genotype. Pharmacogenomics allows a clinician or physicianto target prophylactic or therapeutic treatments to patients who willmost benefit from the treatment and to avoid treatment of patients whowill experience toxic drug-related side effects.

[0251] 1. Prophylactic Methods

[0252] In one aspect, the invention provides a method for preventing ina subject, a disease or condition associated with an aberrant orunwanted PGC-1 expression or activity, by administering to the subject aPGC-1 or an agent which modulates PGC-1 expression or at least one PGC-1activity. Subjects at risk for a disease which is caused or contributedto by aberrant or unwanted PGC-1 expression or activity can beidentified by, for example, any or a combination of diagnostic orprognostic assays as described herein. Administration of a prophylacticagent can occur prior to the manifestation of symptoms characteristic ofthe PGC-1 aberrancy, such that a disease or disorder is prevented or,alternatively, delayed in its progression. Depending on the type ofPGC-1 aberrancy, for example, a PGC-1, PGC-1 agonist or PGC-1 antagonistagent can be used for treating the subject. The appropriate agent can bedetermined based on screening assays described herein.

[0253] 2. Therapeutic Methods

[0254] Another aspect of the invention pertains to methods of modulatingPGC-1 expression or activity for therapeutic purposes. Accordingly, inan exemplary embodiment, the modulatory method of the invention involvescontacting a cell capable of expressing PGC-1 with an agent thatmodulates one or more of the activities of PGC-1 protein activityassociated with the cell, such that PGC-1 activity in the cell ismodulated. An agent that modulates PGC-1 protein activity can be anagent as described herein, such as a nucleic acid or a protein, anaturally-occurring target molecule of a PGC-1 protein (e.g., a PGC-1target molecule), a PGC-1 antibody, a PGC-1 agonist or antagonist, apeptidomimetic of a PGC-1 agonist or antagonist, or other smallmolecule. In one embodiment, the agent stimulates one or more PGC-1activities. Examples of such stimulatory agents include active PGC-1protein and a nucleic acid molecule encoding PGC-1 that has beenintroduced into the cell. In another embodiment, the agent inhibits oneor more PGC-1 activities. Examples of such inhibitory agents includeantisense PGC-1 nucleic acid molecules, anti-PGC-1 antibodies, and PGC-1inhibitors. These modulatory methods can be performed in vitro (e.g., byculturing the cell with the agent) or, alternatively, in vivo (e.g. byadministering the agent to a subject). As such, the present inventionprovides methods of treating an individual afflicted with a disease ordisorder character by aberrant or unwanted expression or activity of aPGC-1 protein or nucleic acid molecule. In one embodiment, the methodinvolves administering an agent (e.g, an agent identified by a screeningassay described herein), or combination of agents that modulates (e.g.,upregulates or downregulates) PGC-1 expression or activity. In anotherembodiment, the method involves administering a PGC-1 protein or nucleicacid molecule as therapy to compensate for reduced, aberrant, orunwanted PGC-1 expression or activity. Stimulation of PGC-1 activity isdesirable in situations in which PGC-1 is abnormally downregulatedand/or in which increased PGC-1 activity is likely to have a beneficialeffect. For example, stimulation of PGC-1 activity is desirable insituations in which a PGC-1 is downregulated and/or in which increasedPGC-1 activity is likely to have a beneficial effect. Likewise,inhibition of PGC-1 activity is desirable in situations in which PGC-1is abnormally upregulated and/or in which decreased PGC-1 activity islikely to have a beneficial effect.

[0255] This invention is further illustrated by the following examples,which should not be construed as limiting. The contents of allreferences, patent applications, patents, and published patentapplications, as well as the Figures and the Sequence Listing, citedthroughout this application are hereby incorporated by reference.

EXAMPLES

[0256] Materials and Methods

[0257] Library Screening and cDNA Cloning

[0258] The PGC-1b and PGC-1c cDNAs were isolated by screening anoligo-dT primed cDNA λZAP library prepared from HIB-1B polyA+ mRNA. Thislibrary was screened using the 297 bp NheI-EcoRI fragment derived fromthe PGC-1a cDNA After three rounds of screening under low stringency(2×SSC, 0.5% SDS at 42° C.), the inserts of thirty-one positive cloneswere excised into pBluescript and sequenced by standard methods. Sevenof the positive clones turned out to be one of two novel isoforms ofPGC-1, referred to herein as PGC-1b and PGC-1c.

[0259] pEGFP-PGC-1b and pEGFP-PGC-1c were constructed by replacing the1866 bp EcoRI-BamHI fragment of pEGFP-PGC-1a with an EcoRI-BamHI PCRfragment corresponding to nucleotide 643-1100 of the PGCI-1b cDNA (SEQID NO:6) or 620-973 of the PGC-1c cDNA (SEQ ID NO:12), respectively. Thevector used was pEGFP-C1 (Clontech).

[0260] To construct adensviral vectors, PCR was used to introduce aBamHI restriction site upstream of the first ATG codon of the PGC-1a andPGC-1b cDNAs. Their respective full length cDNAs were then excised fromthe pEGFP vectors with BamHI and XbaI restriction enzymes and clonedinto pShuttle-CMV cut with BgIII and XbaI. The final adenoviral vectorswere obtained by in vivo-recombination between the pShuttle-CMV-PGC-1aor pShuttleCMV-PGC-1b constructs and the adenovirus backbone, asdescribed in He, T. et al. (1998) Proc. Natl. Acad. Sci. USA95:2509-2514. Adenoviruses were produced in 293 cells and purified bydouble cesium chloride gradient as described in He et al. (1998) supra.

[0261] Cell Culture

[0262] C2C12 myoblast cells were maintained in subconfluent culture inDMEM supplemented with 10% fetal bovine serum (FBS (CellGro)).Differentiation into myotubes was induced when cells reached confluenceby switching the culture to differentiation medium (DMEM containing 2%heat inactivated horse serum (Life Technologies)). Cells were refed withdifferentiation medium daily until complete differentiation was observed(typically by day 4). These cells were then infected overnight withpurified adenoviruses at a multiplicity of infection (MOI) of 500. ThisMOI was sufficient to infect about 90% of the differentiated myotubes,as determined using GFP-expressing adenovirus.

[0263] Immunofluorescence

[0264] 1×10⁶ C2C12 myoblasts were seeded on glass coverslips in 6 wellplates and transfected 18 hours later with 1 μg of expression vectorencoding the indicated GFP fusion protein, using Fugene 6 astransfection reagent (Roche Molecular Biochemicals). 24 to 48 hoursafter transfection, cells were fixed in 4% paraformaldehyde, and thecoverslips were mounted on glass slides and examined by epifluorescence.For catalase staining, cells were permeabilized after fixation with 0.1%Triton-X100 in PBS, blocked in.3% bovine serum albumin (BSA) for 30minutes, and sequentially incubatedwith a sheep polyclonal antibodyraised against human catalase (The Binding Site, Birmingham, U.K) for 1h at 37° C. and a Texas-Red conjugated secondary antibody.

[0265] Respiration Measurements in Intact Cells

[0266] C2C12 myoblasts were seeded in 100 mm dishes, differentiated intomyotubes for 4 days, and infected with the different adenoviruses atequivalent MOIs. Oxygen consumption was measured 48 hours post-infectionas follows: cells were washed with PBS once, mechanically detached fromthe plates using a cell lifter, and centrifuged for 2. min at 500×g, andresuspended in 0.75 ml of differentiation medium.

[0267] 0.5 ml cell suspension was transferred to a 1 ml Clark-typeoxygen electrode chamber (Rank Bros., Bottisham, Cambridge, U.K)maintained at 37° C. and calibrated with air-saturated differentiationmedium. Basal respiration was measured immediately after transfer, forabout 10 minutes. State 4 (non-phosphorylating) respiration was inducedby addition of 320 ng/ml oligomycin (an inhibitor of mitochondrialATPase and phosphoryl group transfer), and the rate measured aftersteady state oxygen consumption was reached. After another 10 minutes, 8μM FCCP (carbonyl cyanide 4-trifluoromethoxyphenylhydrazone, anuncoupler of oxidative phosphorylation) was added to determine uncoupledrespiration in the cells. All respiration rates were expressed relativeto basal respiration as determined at the beginning of each experiment.

[0268] Northern Blotting

[0269] Infected C2C12 myotubes grown in 60 mm dishes were lysed in 1.5ml of Trizol reagent (Life Technologies) and total RNA was purifiedaccording to the manufacturer's instructions. RNA were resolved on 1.2%agarose/formaldehyde gel before transfer to nylon membranes (ICN).Hybridization was performed in UltraHyb solution (Ambion) according tothe manufacturer's instructions. Certain cDNA probes were described inWu, Z. et al. (1999) Cell 98(l):115-24. The following mouse cDNAfragments were used as probes for northern blot analysis: Lipoproteinlipase (LPL; GenBank Accession No. J03302): 400 nucleotide fragment (the400 nucleotides at the 3′ end of the coding region);. FAT/CD36 (GenBankAccession No. L23108): 962 nucleotide fragment (nucleotides 686 to1647); MCD (GenBank Accession No. BC004764): 696 nucleotide fragment(nucleotides 860 to 1555); VLACS (GenBank Accession No. AJ223958): 934nucleotide fragment (nucleotides 864 to.1 797). AOX was detected using a1.4 kb fragment derived from the rat cDNA (GenBank Accession No.J02752). MCAD (GenBank Accession No. NM_(—)007382) was detected usingthe full length cDNA fragment.

Example 1 Identification of Two Novel Isoforms of PGC-1

[0270] PCC-1a was previously identified in a yeast two-hybrid screenaimed at identifying brown fat specific PPARγ transcriptionalco-activators (Puigserver, P. et al. (1998) Cell 92(6):829-39; U.S. Pat.No. 6,166,192; PCT International Publication No.

[0271] WO 98/54220). The nucleotide and amino acid sequences of PGC-1aare shown in SEQ ID NOs:1 and 2, respectively. The coding region ofPGC-1a is shown in SEQ ID NO:3.

[0272] Two shorter PGC-1 have now been identified. These clones,referred to herein as PGC-1b and PGC-1c, contained inserts of 1893 and1744 nucleotides, respectively (SEQ ID NO:6 and SEQ ID NO:12,respectively). PGC-1a, PGC-1b, and PGC-1c are identical through their 5′regions (except for a short region of nucleotides at the extreme 3′ UTRof each isoform; see FIG. 6) but diverge after nucleotide 964 of PGC-1a(SEQ ID NO: 1), corresponding to nucleotide 962 of PGC-1b (SEQ ID NO:6)and nucleotide 939 of PGC-1c (SEQ ID NO:12). See FIGS. 5 and 7. Thiscorresponds to the junction between exon 7 and 8 of the human PGC-1agenomic sequence, suggesting that PGC-1b and PGC-1c could arise fromalternative splicing. PGC-1b and PGC-1c contain predicted open readingframes of 960 and 900 nucleotides, respectively, and therefore lack theC-terminal SR and RRM RNA processing motifs identified in PGC-1a(Monsalve, M. et al. (2000) Mol. Cell 6(2):307-1.6). The proteinsencoded by PGC-1b and PGC-1c are identical to the PGC-1a protein (SEQID:NO.2) through amino acid residues 1-291 of SEQ ID NO:7 (PGC-1b) andSEQ ID NO:13 (PGC-1c), but each contains a unique C-terminal region (seeFIGS. 5 and 7).

Example 2 Expression Pattern of PGC-1b

[0273] To determine the expression pattern of PGC-1b, twooligonucleotides in the 3′ region specific to PGC-1b were designed, andexpression level of PGC-1b was determined by quantitative rt-PCR. Theresults of the rt-PCR analysis demonstrated that PGC-1b is highlyexpressed in heart and hypothalamus, with intermediate levels ofexpression found in kidney, skeletal muscle and brown fat. No signalcould be detected in white fat or spleen. This pattern of expressionclosely overlaps the one described for PGC-1a (Puigserver, P. et al.(1998) Cell 92(6):829-39).

Example 3 PGC-1c dose not Promote Mitochondrial Biogenesis or Function

[0274] PGC-1a promotes mitochondrial biogenesis and respiration in C2C12cells (Wu, Z. et al. (1999) Cell 98(1):115-24). In order to determinewhether PGC-1b can also do so, C2C12 myoblasts (at day five ofdifferentiation) were infected with adenoviruses expressing either GFP(as a control), PGC-1a, or PGC-1b. Oxygen consumption was measured 48hours after infection. While PGC-1a stimulated both total andoligomycin-dependent respiration, as well as overall mitochondrialcapacity (as assessed by the O2 consumption in the presence of theuncoupler FCCP), PGC-1b expression did not significantly affect any ofthese variables.

[0275] The effect of PGC-1a on mitochondrial respiration has been linkedto its ability to induce components of the respiratory chain (B ATPsynthetase, CytC, CoxII and CoxIV). In agreement with its lack of effecton mitochondrial respiration, PGC-1b did not stimulate the expression ofany of the respiratory chain genes, nor did it induce the mRNA for mTFAor NRF-1, two key factors involved in mitochondrial biogenesis.

[0276] Since PGC-1a orchestrates mitochondrial biogenesis and theinduction of several respiratory genes by co-activating NRF-1 (Wu, Z. etal. (1999) Cell 98(1):115-24), the ability of PGC-1b to co-activate thistranscription factor in transient transfection was tested. However,despite the fact that PGC-1b contains the N-terminal transcriptionactivation domain and most of the transcription factor interactionmotifs present in PGC-1a (Puigserver, P. et al. (1999) Science286(5443):1368-71), it failed to activate the NRF-1 reporter gene.

[0277] The fact that PGC-1b appears to be, based on the data above, aninactive variant of the full length PGC-1a, prompted an evaluation ofwhether it could act as a dominant negative regulator of PGC-1a. C2C12myoblasts were infected with a constant MOI for PGC-1-a adenovirus andan increasing MOI of GFP (as a control) or PGC-1b-viruses. Increasingthe expression of PGC-1b did not prevent the induction of Cytochrome C,a direct target of the NRF-1/PGC-1a complex.

[0278] Taken together, these results demonstrate that PGC-1b does notinfluence mitochondrial biogenesis or function, either directly orthrough interference with PGC-1a, and suggest that PGC-1b has a specificfunction distinct from the role of PGC-1a.

Example 4 PGC-1b is Localized to Peroxisomes

[0279] The inability of PGC-1b to activate known PGC-1a target genes,despite the presence of both the N-terminal activation domain and mostof the transcription factor interaction motifs prompted examination ofits subcellular localization. A GFP-PGC-1b fusion protein was localizedto the cytoplasm and showed a striking punctate pattern, in contrastwith the nuclear and cytoplasmic localization observed for GFP-PGC-1aand GFP-PGC-1c, respectively. Because PGC-1b and PGC-1c differ only intheir unique C-termini, it was hypothesized that the unique C-terminusof PGC-1b might dictate its punctate localization within the cytoplasm.To test this hypothesis directly, the C-terminal 29 amino acids ofPGC-1b (the unique region, corresponding to amino acid residues 292-320of SEQ ID NO:7, set forth as SEQ ID NO:10) were fused to GFP. Theresulting fusion protein localized to cytoplasmic dots, demonstratingthat these 29 amino acids are sufficient to direct the punctatelocalization of PGC-1b.

[0280] Analysis of the PGC-1b unique C-terminal domain revealed thepresence of a carboxy-terminal tripeptide (at residues 318-320 of SEQ IDNO:7 and residues 27-29 of SEQ ID NO:10) resembling the peroxisomallocalization signal found in the enzyme d-aspartate oxidase(Ser-Asn-Leu-COOH; Amery, L. et al. (1998) Biochem. J. 336(Pt.2):367-71).

[0281] To determine if the punctate localization corresponded toperoxisomes, cells transfected with GFP-PGC-1b were stained with anantibody specific for the peroxisomal marker catalase. The results ofthis analysis indicated that the GFP-PGC-1b and catalase signalscolocalized, demonstrating that PGC-1b is indeed targeted toperoxisomes. Finally, using an antibody specific for the amino-terminalregion of PGC-1, the peroxisomal localization was confirmed with annon-tagged form of PGC-1b. This analysis further showed that PGC-1bremained localized to peroxisomes in fully differentiated C2C12myotubes.

Example 5 PGC-1b Induces Genes Involved in Fatty-Acid Uptake andOxidation

[0282] The peroxisomal localization of PGC-1b prompted examination ofits potential involvement as a regulator of fatty-acid oxidation genes.The expression of PGC-1b in fully differentiated C2C12 myotubes induceda panel of key regulatory genes along the fatty-acid metabolic pathway:lipoprotein lipase (LPL), which hydrolyses the core triglycerides ofcirculating chylomicron; fatty acid translocase (FAT/CD36), which ispart of a specialized, protein-facilitated membrane transport system forlong chain fatty-acids; very long chain acyl-CoA synthetase (VLACS), aperoxisomal membrane protein involved in the import of very long chainfatty acids in peroxisomes; acyl Co-A oxidase (AOX), the rate limitingenzyme in peroxisomal fatty acid oxidation; medium chain acyl-CoAdehydrogenase (MCAD), which catalyzes a pivotal step in mitochondrialfatty acid oxidation; and malonyl-CoA dehydrogenase (MCD), whose actionlowers the steady state level of malonyl-CoA, an allosteric inhibitor ofcarnitine palmitoyltransferase-1 (CPT-1), an enzyme which promotes fattyacid oxidation. Some of these genes were specifically induced by PGC-1b(LPL and AOX), while others were activated by both PGC-1b and PGC-1a(FAT/CD36, VLACS, MCD and MCAD).

[0283] Despite the role of PPARα in the induction of most of these genesin the liver, and the fact that PGC-1a coactivates PPARα (Vega, R. B. etal. (2000) Mol. Cell. Biol. 20(5):1868-76), treatment with the PPARαligand WY, even at saturating doses, did not result in any furtherstimulation of their expression. These results were confirmed in C2C12myoblasts which overexpressed PPARα. Although treatment with WY ligandcould induce some PPARα targets (e.g., UCP3), no cooperativity wasobserved between PGC-1b-mediated gene activation and PPARα, suggestingthat in C2C12 cells, PGC-1b activates AOX and LPL in a PPARα independentmanner.

[0284] Equivalents

[0285] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein Such equivalentsare intended to-be encompassed by the following claims.

1 17 1 3066 DNA Mus musculus CDS (92)...(2482) 1 aattcggcac gaggttgcctgcatgagtgt gtgctgtgtg tcagagtgga ttggagttga 60 aaaagcttga ctggcgtcattcgggagctg g atg gct tgg gac atg tgc agc 112 Met Ala Trp Asp Met Cys Ser1 5 caa gac tct gta tgg agt gac ata gag tgt gct gct ctg gtt ggt gag 160Gln Asp Ser Val Trp Ser Asp Ile Glu Cys Ala Ala Leu Val Gly Glu 10 15 20gac cag cct ctt tgc cca gat ctt cct gaa ctt gac ctt tct gaa ctt 208 AspGln Pro Leu Cys Pro Asp Leu Pro Glu Leu Asp Leu Ser Glu Leu 25 30 35 gatgtg aat gac ttg gat aca gac agc ttt ctg ggt gga ttg aag tgg 256 Asp ValAsn Asp Leu Asp Thr Asp Ser Phe Leu Gly Gly Leu Lys Trp 40 45 50 55 tgtagc gac caa tcg gaa atc ata tcc aac cag tac aac aat gag cct 304 Cys SerAsp Gln Ser Glu Ile Ile Ser Asn Gln Tyr Asn Asn Glu Pro 60 65 70 gcg aacata ttt gag aag ata gat gaa gag aat gag gca aac ttg cta 352 Ala Asn IlePhe Glu Lys Ile Asp Glu Glu Asn Glu Ala Asn Leu Leu 75 80 85 gcg gtc ctcaca gag aca ctg gac agt ctc ccc gtg gat gaa gac gga 400 Ala Val Leu ThrGlu Thr Leu Asp Ser Leu Pro Val Asp Glu Asp Gly 90 95 100 ttg ccc tcattt gat gca ctg aca gat gga gcc gtg acc act gac aac 448 Leu Pro Ser PheAsp Ala Leu Thr Asp Gly Ala Val Thr Thr Asp Asn 105 110 115 gag gcc agtcct tcc tcc atg cct gac ggc acc cct ccc cct cag gag 496 Glu Ala Ser ProSer Ser Met Pro Asp Gly Thr Pro Pro Pro Gln Glu 120 125 130 135 gca gaagag ccg tct cta ctt aag aag ctc tta ctg gca cca gcc aac 544 Ala Glu GluPro Ser Leu Leu Lys Lys Leu Leu Leu Ala Pro Ala Asn 140 145 150 act cagctc agc tac aat gaa tgc agc ggt ctt agc act cag aac cat 592 Thr Gln LeuSer Tyr Asn Glu Cys Ser Gly Leu Ser Thr Gln Asn His 155 160 165 gca gcaaac cac acc cac agg atc aga aca aac cct gcc att gtt aag 640 Ala Ala AsnHis Thr His Arg Ile Arg Thr Asn Pro Ala Ile Val Lys 170 175 180 acc gagaat tca tgg agc aat aaa gcg aag agc att tgt caa cag caa 688 Thr Glu AsnSer Trp Ser Asn Lys Ala Lys Ser Ile Cys Gln Gln Gln 185 190 195 aag ccacaa aga cgt ccc tgc tca gag ctt ctc aag tat ctg acc aca 736 Lys Pro GlnArg Arg Pro Cys Ser Glu Leu Leu Lys Tyr Leu Thr Thr 200 205 210 215 aacgat gac cct cct cac acc aaa ccc aca gaa aac agg aac agc agc 784 Asn AspAsp Pro Pro His Thr Lys Pro Thr Glu Asn Arg Asn Ser Ser 220 225 230 agagac aaa tgt gct tcc aaa aag aag tcc cat aca caa ccg cag tcg 832 Arg AspLys Cys Ala Ser Lys Lys Lys Ser His Thr Gln Pro Gln Ser 235 240 245 caacat gct caa gcc aaa cca aca act tta tct ctt cct ctg acc cca 880 Gln HisAla Gln Ala Lys Pro Thr Thr Leu Ser Leu Pro Leu Thr Pro 250 255 260 gagtca cca aat gac ccc aag ggt tcc cca ttt gag aac aag act att 928 Glu SerPro Asn Asp Pro Lys Gly Ser Pro Phe Glu Asn Lys Thr Ile 265 270 275 gagcga acc tta agt gtg gaa ctc tct gga act gca ggc cta act cct 976 Glu ArgThr Leu Ser Val Glu Leu Ser Gly Thr Ala Gly Leu Thr Pro 280 285 290 295ccc aca act cct cct cat aaa gcc aac caa gat aac cct ttc aag gct 1024 ProThr Thr Pro Pro His Lys Ala Asn Gln Asp Asn Pro Phe Lys Ala 300 305 310tcg cca aag ctg aag ccc tct tgc aag acc gtg gtg cca ccg cca acc 1072 SerPro Lys Leu Lys Pro Ser Cys Lys Thr Val Val Pro Pro Pro Thr 315 320 325aag agg gcc cgg tac agt gag tgt tct ggt acc caa ggc agc cac tcc 1120 LysArg Ala Arg Tyr Ser Glu Cys Ser Gly Thr Gln Gly Ser His Ser 330 335 340acc aag aaa ggg ccc gag caa tct gag ttg tac gca caa ctc agc aag 1168 ThrLys Lys Gly Pro Glu Gln Ser Glu Leu Tyr Ala Gln Leu Ser Lys 345 350 355tcc tca ggg ctc agc cga gga cac gag gaa agg aag act aaa cgg ccc 1216 SerSer Gly Leu Ser Arg Gly His Glu Glu Arg Lys Thr Lys Arg Pro 360 365 370375 agt ctc cgg ctg ttt ggt gac cat gac tac tgt cag tca ctc aat tcc 1264Ser Leu Arg Leu Phe Gly Asp His Asp Tyr Cys Gln Ser Leu Asn Ser 380 385390 aaa acg gat ata ctc att aac ata tca cag gag ctc caa gac tct aga 1312Lys Thr Asp Ile Leu Ile Asn Ile Ser Gln Glu Leu Gln Asp Ser Arg 395 400405 caa cta gac ttc aaa gat gcc tcc tgt gac tgg cag ggg cac atc tgt 1360Gln Leu Asp Phe Lys Asp Ala Ser Cys Asp Trp Gln Gly His Ile Cys 410 415420 tct tcc aca gat tca ggc cag tgc tac ctg aga gag act ttg gag gcc 1408Ser Ser Thr Asp Ser Gly Gln Cys Tyr Leu Arg Glu Thr Leu Glu Ala 425 430435 agc aag cag gtc tct cct tgc agc acc aga aaa cag ctc caa gac cag 1456Ser Lys Gln Val Ser Pro Cys Ser Thr Arg Lys Gln Leu Gln Asp Gln 440 445450 455 gaa atc cga gcg gag ctg aac aag cac ttc ggt cat ccc tgt caa gct1504 Glu Ile Arg Ala Glu Leu Asn Lys His Phe Gly His Pro Cys Gln Ala 460465 470 gtg ttt gac gac aaa tca gac aag acc agt gaa cta agg gat ggc gac1552 Val Phe Asp Asp Lys Ser Asp Lys Thr Ser Glu Leu Arg Asp Gly Asp 475480 485 ttc agt aat gaa caa ttc tcc aaa cta cct gtg ttt ata aat tca gga1600 Phe Ser Asn Glu Gln Phe Ser Lys Leu Pro Val Phe Ile Asn Ser Gly 490495 500 cta gcc atg gat ggc cta ttt gat gac agt gaa gat gaa agt gat aaa1648 Leu Ala Met Asp Gly Leu Phe Asp Asp Ser Glu Asp Glu Ser Asp Lys 505510 515 ctg agc tac cct tgg gat ggc acg cag ccc tat tca ttg ttc gat gtg1696 Leu Ser Tyr Pro Trp Asp Gly Thr Gln Pro Tyr Ser Leu Phe Asp Val 520525 530 535 tcg cct tct tgc tct tcc ttt aac tct ccg tgt cga gac tca gtgtca 1744 Ser Pro Ser Cys Ser Ser Phe Asn Ser Pro Cys Arg Asp Ser Val Ser540 545 550 cca ccg aaa tcc tta ttt tct caa aga ccc caa agg atg cgc tctcgt 1792 Pro Pro Lys Ser Leu Phe Ser Gln Arg Pro Gln Arg Met Arg Ser Arg555 560 565 tca aga tcc ttt tct cga cac agg tcg tgt tcc cga tca cca tattcc 1840 Ser Arg Ser Phe Ser Arg His Arg Ser Cys Ser Arg Ser Pro Tyr Ser570 575 580 agg tca aga tca agg tcc cca ggc agt aga tcc tct tca aga tcctgt 1888 Arg Ser Arg Ser Arg Ser Pro Gly Ser Arg Ser Ser Ser Arg Ser Cys585 590 595 tac tac tat gaa tca agc cac tac aga cac cgc aca cac cgc aattct 1936 Tyr Tyr Tyr Glu Ser Ser His Tyr Arg His Arg Thr His Arg Asn Ser600 605 610 615 ccc ttg tat gtg aga tca cgt tca agg tca ccc tac agc cgtagg ccc 1984 Pro Leu Tyr Val Arg Ser Arg Ser Arg Ser Pro Tyr Ser Arg ArgPro 620 625 630 agg tac gac agc tat gaa gcc tat gag cac gaa agg ctc aagagg gat 2032 Arg Tyr Asp Ser Tyr Glu Ala Tyr Glu His Glu Arg Leu Lys ArgAsp 635 640 645 gaa tac cgc aaa gag cac gag aag cgg gag tct gaa agg gccaaa cag 2080 Glu Tyr Arg Lys Glu His Glu Lys Arg Glu Ser Glu Arg Ala LysGln 650 655 660 aga gag agg cag aag cag aaa gca att gaa gag cgc cgt gtgatt tac 2128 Arg Glu Arg Gln Lys Gln Lys Ala Ile Glu Glu Arg Arg Val IleTyr 665 670 675 gtt ggt aaa atc aga cct gac aca acg cgg aca gaa ttg agagac cgc 2176 Val Gly Lys Ile Arg Pro Asp Thr Thr Arg Thr Glu Leu Arg AspArg 680 685 690 695 ttt gaa gtt ttt ggt gaa att gag gaa tgc acc gta aatctg cgg gat 2224 Phe Glu Val Phe Gly Glu Ile Glu Glu Cys Thr Val Asn LeuArg Asp 700 705 710 gat gga gac agc tat ggt ttc atc acc tac cgt tac acctgt gac gct 2272 Asp Gly Asp Ser Tyr Gly Phe Ile Thr Tyr Arg Tyr Thr CysAsp Ala 715 720 725 ttc gct gct ctt gag aat gga tat act tta cgc agg tcgaac gaa act 2320 Phe Ala Ala Leu Glu Asn Gly Tyr Thr Leu Arg Arg Ser AsnGlu Thr 730 735 740 gac ttc gag ctg tac ttt tgt gga cgg aag caa ttt ttcaag tct aac 2368 Asp Phe Glu Leu Tyr Phe Cys Gly Arg Lys Gln Phe Phe LysSer Asn 745 750 755 tat gca gac cta gat acc aac tca gac gat ttt gac cctgct tcc acc 2416 Tyr Ala Asp Leu Asp Thr Asn Ser Asp Asp Phe Asp Pro AlaSer Thr 760 765 770 775 aag agc aag tat gac tct ctg gat ttt gat agt ttactg aag gaa gct 2464 Lys Ser Lys Tyr Asp Ser Leu Asp Phe Asp Ser Leu LeuLys Glu Ala 780 785 790 cag aga agc ttg cgc agg taacgtgttc ccaggctgaggaatgacaga 2512 Gln Arg Ser Leu Arg Arg 795 gagatggtca atacctcatgggacagcgtg tcctttccca agactcttgc aagtcatact 2572 taggaatttc tcctactttacactctctgt acaaaaataa aacaaaacaa aacaacaata 2632 acaacaacaa caacaacaataacaacaaca accataccag aacaagaaca acggtttaca 2692 tgaacacagc tgctgaagaggcaagagaca gaatgataat ccagtaagca cacgtttatt 2752 cacgggtgtc agctttgctttccctggagg ctcttggtga cagtgtgtgt gcgtgtgtgt 2812 gtgtgggtgt gcgtgtgtgtatgtgtgtgt gtgtacttgt ttggaaagta catatgtaca 2872 catgtgagga cttgggggcacctgaacaga acgaacaagg gcgacccctt caaatggcag 2932 catttccatg aagacacacttaaaacctac aacttcaaaa tgttcgtatt ctatacaaaa 2992 ggaaaataaa taaatataaaaaaaaaaaaa aaaaaactcg agagatctat gaatcgtaga 3052 tactgaaaaa cccc 3066 2797 PRT Mus musculus 2 Met Ala Trp Asp Met Cys Ser Gln Asp Ser Val TrpSer Asp Ile Glu 1 5 10 15 Cys Ala Ala Leu Val Gly Glu Asp Gln Pro LeuCys Pro Asp Leu Pro 20 25 30 Glu Leu Asp Leu Ser Glu Leu Asp Val Asn AspLeu Asp Thr Asp Ser 35 40 45 Phe Leu Gly Gly Leu Lys Trp Cys Ser Asp GlnSer Glu Ile Ile Ser 50 55 60 Asn Gln Tyr Asn Asn Glu Pro Ala Asn Ile PheGlu Lys Ile Asp Glu 65 70 75 80 Glu Asn Glu Ala Asn Leu Leu Ala Val LeuThr Glu Thr Leu Asp Ser 85 90 95 Leu Pro Val Asp Glu Asp Gly Leu Pro SerPhe Asp Ala Leu Thr Asp 100 105 110 Gly Ala Val Thr Thr Asp Asn Glu AlaSer Pro Ser Ser Met Pro Asp 115 120 125 Gly Thr Pro Pro Pro Gln Glu AlaGlu Glu Pro Ser Leu Leu Lys Lys 130 135 140 Leu Leu Leu Ala Pro Ala AsnThr Gln Leu Ser Tyr Asn Glu Cys Ser 145 150 155 160 Gly Leu Ser Thr GlnAsn His Ala Ala Asn His Thr His Arg Ile Arg 165 170 175 Thr Asn Pro AlaIle Val Lys Thr Glu Asn Ser Trp Ser Asn Lys Ala 180 185 190 Lys Ser IleCys Gln Gln Gln Lys Pro Gln Arg Arg Pro Cys Ser Glu 195 200 205 Leu LeuLys Tyr Leu Thr Thr Asn Asp Asp Pro Pro His Thr Lys Pro 210 215 220 ThrGlu Asn Arg Asn Ser Ser Arg Asp Lys Cys Ala Ser Lys Lys Lys 225 230 235240 Ser His Thr Gln Pro Gln Ser Gln His Ala Gln Ala Lys Pro Thr Thr 245250 255 Leu Ser Leu Pro Leu Thr Pro Glu Ser Pro Asn Asp Pro Lys Gly Ser260 265 270 Pro Phe Glu Asn Lys Thr Ile Glu Arg Thr Leu Ser Val Glu LeuSer 275 280 285 Gly Thr Ala Gly Leu Thr Pro Pro Thr Thr Pro Pro His LysAla Asn 290 295 300 Gln Asp Asn Pro Phe Lys Ala Ser Pro Lys Leu Lys ProSer Cys Lys 305 310 315 320 Thr Val Val Pro Pro Pro Thr Lys Arg Ala ArgTyr Ser Glu Cys Ser 325 330 335 Gly Thr Gln Gly Ser His Ser Thr Lys LysGly Pro Glu Gln Ser Glu 340 345 350 Leu Tyr Ala Gln Leu Ser Lys Ser SerGly Leu Ser Arg Gly His Glu 355 360 365 Glu Arg Lys Thr Lys Arg Pro SerLeu Arg Leu Phe Gly Asp His Asp 370 375 380 Tyr Cys Gln Ser Leu Asn SerLys Thr Asp Ile Leu Ile Asn Ile Ser 385 390 395 400 Gln Glu Leu Gln AspSer Arg Gln Leu Asp Phe Lys Asp Ala Ser Cys 405 410 415 Asp Trp Gln GlyHis Ile Cys Ser Ser Thr Asp Ser Gly Gln Cys Tyr 420 425 430 Leu Arg GluThr Leu Glu Ala Ser Lys Gln Val Ser Pro Cys Ser Thr 435 440 445 Arg LysGln Leu Gln Asp Gln Glu Ile Arg Ala Glu Leu Asn Lys His 450 455 460 PheGly His Pro Cys Gln Ala Val Phe Asp Asp Lys Ser Asp Lys Thr 465 470 475480 Ser Glu Leu Arg Asp Gly Asp Phe Ser Asn Glu Gln Phe Ser Lys Leu 485490 495 Pro Val Phe Ile Asn Ser Gly Leu Ala Met Asp Gly Leu Phe Asp Asp500 505 510 Ser Glu Asp Glu Ser Asp Lys Leu Ser Tyr Pro Trp Asp Gly ThrGln 515 520 525 Pro Tyr Ser Leu Phe Asp Val Ser Pro Ser Cys Ser Ser PheAsn Ser 530 535 540 Pro Cys Arg Asp Ser Val Ser Pro Pro Lys Ser Leu PheSer Gln Arg 545 550 555 560 Pro Gln Arg Met Arg Ser Arg Ser Arg Ser PheSer Arg His Arg Ser 565 570 575 Cys Ser Arg Ser Pro Tyr Ser Arg Ser ArgSer Arg Ser Pro Gly Ser 580 585 590 Arg Ser Ser Ser Arg Ser Cys Tyr TyrTyr Glu Ser Ser His Tyr Arg 595 600 605 His Arg Thr His Arg Asn Ser ProLeu Tyr Val Arg Ser Arg Ser Arg 610 615 620 Ser Pro Tyr Ser Arg Arg ProArg Tyr Asp Ser Tyr Glu Ala Tyr Glu 625 630 635 640 His Glu Arg Leu LysArg Asp Glu Tyr Arg Lys Glu His Glu Lys Arg 645 650 655 Glu Ser Glu ArgAla Lys Gln Arg Glu Arg Gln Lys Gln Lys Ala Ile 660 665 670 Glu Glu ArgArg Val Ile Tyr Val Gly Lys Ile Arg Pro Asp Thr Thr 675 680 685 Arg ThrGlu Leu Arg Asp Arg Phe Glu Val Phe Gly Glu Ile Glu Glu 690 695 700 CysThr Val Asn Leu Arg Asp Asp Gly Asp Ser Tyr Gly Phe Ile Thr 705 710 715720 Tyr Arg Tyr Thr Cys Asp Ala Phe Ala Ala Leu Glu Asn Gly Tyr Thr 725730 735 Leu Arg Arg Ser Asn Glu Thr Asp Phe Glu Leu Tyr Phe Cys Gly Arg740 745 750 Lys Gln Phe Phe Lys Ser Asn Tyr Ala Asp Leu Asp Thr Asn SerAsp 755 760 765 Asp Phe Asp Pro Ala Ser Thr Lys Ser Lys Tyr Asp Ser LeuAsp Phe 770 775 780 Asp Ser Leu Leu Lys Glu Ala Gln Arg Ser Leu Arg Arg785 790 795 3 2391 DNA Mus musculus CDS (1)...(2391) 3 atg gct tgg gacatg tgc agc caa gac tct gta tgg agt gac ata gag 48 Met Ala Trp Asp MetCys Ser Gln Asp Ser Val Trp Ser Asp Ile Glu 1 5 10 15 tgt gct gct ctggtt ggt gag gac cag cct ctt tgc cca gat ctt cct 96 Cys Ala Ala Leu ValGly Glu Asp Gln Pro Leu Cys Pro Asp Leu Pro 20 25 30 gaa ctt gac ctt tctgaa ctt gat gtg aat gac ttg gat aca gac agc 144 Glu Leu Asp Leu Ser GluLeu Asp Val Asn Asp Leu Asp Thr Asp Ser 35 40 45 ttt ctg ggt gga ttg aagtgg tgt agc gac caa tcg gaa atc ata tcc 192 Phe Leu Gly Gly Leu Lys TrpCys Ser Asp Gln Ser Glu Ile Ile Ser 50 55 60 aac cag tac aac aat gag cctgcg aac ata ttt gag aag ata gat gaa 240 Asn Gln Tyr Asn Asn Glu Pro AlaAsn Ile Phe Glu Lys Ile Asp Glu 65 70 75 80 gag aat gag gca aac ttg ctagcg gtc ctc aca gag aca ctg gac agt 288 Glu Asn Glu Ala Asn Leu Leu AlaVal Leu Thr Glu Thr Leu Asp Ser 85 90 95 ctc ccc gtg gat gaa gac gga ttgccc tca ttt gat gca ctg aca gat 336 Leu Pro Val Asp Glu Asp Gly Leu ProSer Phe Asp Ala Leu Thr Asp 100 105 110 gga gcc gtg acc act gac aac gaggcc agt cct tcc tcc atg cct gac 384 Gly Ala Val Thr Thr Asp Asn Glu AlaSer Pro Ser Ser Met Pro Asp 115 120 125 ggc acc cct ccc cct cag gag gcagaa gag ccg tct cta ctt aag aag 432 Gly Thr Pro Pro Pro Gln Glu Ala GluGlu Pro Ser Leu Leu Lys Lys 130 135 140 ctc tta ctg gca cca gcc aac actcag ctc agc tac aat gaa tgc agc 480 Leu Leu Leu Ala Pro Ala Asn Thr GlnLeu Ser Tyr Asn Glu Cys Ser 145 150 155 160 ggt ctt agc act cag aac catgca gca aac cac acc cac agg atc aga 528 Gly Leu Ser Thr Gln Asn His AlaAla Asn His Thr His Arg Ile Arg 165 170 175 aca aac cct gcc att gtt aagacc gag aat tca tgg agc aat aaa gcg 576 Thr Asn Pro Ala Ile Val Lys ThrGlu Asn Ser Trp Ser Asn Lys Ala 180 185 190 aag agc att tgt caa cag caaaag cca caa aga cgt ccc tgc tca gag 624 Lys Ser Ile Cys Gln Gln Gln LysPro Gln Arg Arg Pro Cys Ser Glu 195 200 205 ctt ctc aag tat ctg acc acaaac gat gac cct cct cac acc aaa ccc 672 Leu Leu Lys Tyr Leu Thr Thr AsnAsp Asp Pro Pro His Thr Lys Pro 210 215 220 aca gaa aac agg aac agc agcaga gac aaa tgt gct tcc aaa aag aag 720 Thr Glu Asn Arg Asn Ser Ser ArgAsp Lys Cys Ala Ser Lys Lys Lys 225 230 235 240 tcc cat aca caa ccg cagtcg caa cat gct caa gcc aaa cca aca act 768 Ser His Thr Gln Pro Gln SerGln His Ala Gln Ala Lys Pro Thr Thr 245 250 255 tta tct ctt cct ctg acccca gag tca cca aat gac ccc aag ggt tcc 816 Leu Ser Leu Pro Leu Thr ProGlu Ser Pro Asn Asp Pro Lys Gly Ser 260 265 270 cca ttt gag aac aag actatt gag cga acc tta agt gtg gaa ctc tct 864 Pro Phe Glu Asn Lys Thr IleGlu Arg Thr Leu Ser Val Glu Leu Ser 275 280 285 gga act gca ggc cta actcct ccc aca act cct cct cat aaa gcc aac 912 Gly Thr Ala Gly Leu Thr ProPro Thr Thr Pro Pro His Lys Ala Asn 290 295 300 caa gat aac cct ttc aaggct tcg cca aag ctg aag ccc tct tgc aag 960 Gln Asp Asn Pro Phe Lys AlaSer Pro Lys Leu Lys Pro Ser Cys Lys 305 310 315 320 acc gtg gtg cca ccgcca acc aag agg gcc cgg tac agt gag tgt tct 1008 Thr Val Val Pro Pro ProThr Lys Arg Ala Arg Tyr Ser Glu Cys Ser 325 330 335 ggt acc caa ggc agccac tcc acc aag aaa ggg ccc gag caa tct gag 1056 Gly Thr Gln Gly Ser HisSer Thr Lys Lys Gly Pro Glu Gln Ser Glu 340 345 350 ttg tac gca caa ctcagc aag tcc tca ggg ctc agc cga gga cac gag 1104 Leu Tyr Ala Gln Leu SerLys Ser Ser Gly Leu Ser Arg Gly His Glu 355 360 365 gaa agg aag act aaacgg ccc agt ctc cgg ctg ttt ggt gac cat gac 1152 Glu Arg Lys Thr Lys ArgPro Ser Leu Arg Leu Phe Gly Asp His Asp 370 375 380 tac tgt cag tca ctcaat tcc aaa acg gat ata ctc att aac ata tca 1200 Tyr Cys Gln Ser Leu AsnSer Lys Thr Asp Ile Leu Ile Asn Ile Ser 385 390 395 400 cag gag ctc caagac tct aga caa cta gac ttc aaa gat gcc tcc tgt 1248 Gln Glu Leu Gln AspSer Arg Gln Leu Asp Phe Lys Asp Ala Ser Cys 405 410 415 gac tgg cag gggcac atc tgt tct tcc aca gat tca ggc cag tgc tac 1296 Asp Trp Gln Gly HisIle Cys Ser Ser Thr Asp Ser Gly Gln Cys Tyr 420 425 430 ctg aga gag actttg gag gcc agc aag cag gtc tct cct tgc agc acc 1344 Leu Arg Glu Thr LeuGlu Ala Ser Lys Gln Val Ser Pro Cys Ser Thr 435 440 445 aga aaa cag ctccaa gac cag gaa atc cga gcg gag ctg aac aag cac 1392 Arg Lys Gln Leu GlnAsp Gln Glu Ile Arg Ala Glu Leu Asn Lys His 450 455 460 ttc ggt cat ccctgt caa gct gtg ttt gac gac aaa tca gac aag acc 1440 Phe Gly His Pro CysGln Ala Val Phe Asp Asp Lys Ser Asp Lys Thr 465 470 475 480 agt gaa ctaagg gat ggc gac ttc agt aat gaa caa ttc tcc aaa cta 1488 Ser Glu Leu ArgAsp Gly Asp Phe Ser Asn Glu Gln Phe Ser Lys Leu 485 490 495 cct gtg tttata aat tca gga cta gcc atg gat ggc cta ttt gat gac 1536 Pro Val Phe IleAsn Ser Gly Leu Ala Met Asp Gly Leu Phe Asp Asp 500 505 510 agt gaa gatgaa agt gat aaa ctg agc tac cct tgg gat ggc acg cag 1584 Ser Glu Asp GluSer Asp Lys Leu Ser Tyr Pro Trp Asp Gly Thr Gln 515 520 525 ccc tat tcattg ttc gat gtg tcg cct tct tgc tct tcc ttt aac tct 1632 Pro Tyr Ser LeuPhe Asp Val Ser Pro Ser Cys Ser Ser Phe Asn Ser 530 535 540 ccg tgt cgagac tca gtg tca cca ccg aaa tcc tta ttt tct caa aga 1680 Pro Cys Arg AspSer Val Ser Pro Pro Lys Ser Leu Phe Ser Gln Arg 545 550 555 560 ccc caaagg atg cgc tct cgt tca aga tcc ttt tct cga cac agg tcg 1728 Pro Gln ArgMet Arg Ser Arg Ser Arg Ser Phe Ser Arg His Arg Ser 565 570 575 tgt tcccga tca cca tat tcc agg tca aga tca agg tcc cca ggc agt 1776 Cys Ser ArgSer Pro Tyr Ser Arg Ser Arg Ser Arg Ser Pro Gly Ser 580 585 590 aga tcctct tca aga tcc tgt tac tac tat gaa tca agc cac tac aga 1824 Arg Ser SerSer Arg Ser Cys Tyr Tyr Tyr Glu Ser Ser His Tyr Arg 595 600 605 cac cgcaca cac cgc aat tct ccc ttg tat gtg aga tca cgt tca agg 1872 His Arg ThrHis Arg Asn Ser Pro Leu Tyr Val Arg Ser Arg Ser Arg 610 615 620 tca ccctac agc cgt agg ccc agg tac gac agc tat gaa gcc tat gag 1920 Ser Pro TyrSer Arg Arg Pro Arg Tyr Asp Ser Tyr Glu Ala Tyr Glu 625 630 635 640 cacgaa agg ctc aag agg gat gaa tac cgc aaa gag cac gag aag cgg 1968 His GluArg Leu Lys Arg Asp Glu Tyr Arg Lys Glu His Glu Lys Arg 645 650 655 gagtct gaa agg gcc aaa cag aga gag agg cag aag cag aaa gca att 2016 Glu SerGlu Arg Ala Lys Gln Arg Glu Arg Gln Lys Gln Lys Ala Ile 660 665 670 gaagag cgc cgt gtg att tac gtt ggt aaa atc aga cct gac aca acg 2064 Glu GluArg Arg Val Ile Tyr Val Gly Lys Ile Arg Pro Asp Thr Thr 675 680 685 cggaca gaa ttg aga gac cgc ttt gaa gtt ttt ggt gaa att gag gaa 2112 Arg ThrGlu Leu Arg Asp Arg Phe Glu Val Phe Gly Glu Ile Glu Glu 690 695 700 tgcacc gta aat ctg cgg gat gat gga gac agc tat ggt ttc atc acc 2160 Cys ThrVal Asn Leu Arg Asp Asp Gly Asp Ser Tyr Gly Phe Ile Thr 705 710 715 720tac cgt tac acc tgt gac gct ttc gct gct ctt gag aat gga tat act 2208 TyrArg Tyr Thr Cys Asp Ala Phe Ala Ala Leu Glu Asn Gly Tyr Thr 725 730 735tta cgc agg tcg aac gaa act gac ttc gag ctg tac ttt tgt gga cgg 2256 LeuArg Arg Ser Asn Glu Thr Asp Phe Glu Leu Tyr Phe Cys Gly Arg 740 745 750aag caa ttt ttc aag tct aac tat gca gac cta gat acc aac tca gac 2304 LysGln Phe Phe Lys Ser Asn Tyr Ala Asp Leu Asp Thr Asn Ser Asp 755 760 765gat ttt gac cct gct tcc acc aag agc aag tat gac tct ctg gat ttt 2352 AspPhe Asp Pro Ala Ser Thr Lys Ser Lys Tyr Asp Ser Leu Asp Phe 770 775 780gat agt tta ctg aag gaa gct cag aga agc ttg cgc agg 2391 Asp Ser Leu LeuLys Glu Ala Gln Arg Ser Leu Arg Arg 785 790 795 4 924 DNA Mus musculus 4tcagagtgga ttggagttga aaaagcttga ctggcgtcat tcgggagctg gatggcttgg 60gacatgtgca gccaagactc tgtatggagt gacatagagt gtgctgctct ggttggtgag 120gaccagcctc tttgcccaga tcttcctgaa cttgaccttt ctgaacttga tgtgaatgac 180ttggatacag acagctttct gggtggattg aagtggtgta gcgaccaatc ggaaatcata 240tccaaccagt acaacaatga gcctgcgaac atatttgaga agatagatga agagaatgag 300gcgaacttgc tagcggtcct cacagagaca ctggacagtc tccccgtgga tgaagacgga 360ttgccctcat ttgatgcact gacagatgga gccgtgacca ctgacaacga ggccagtcct 420tcctccatgc ctgacggcac ccctccccct caggaggcag aagagccgtc tctacttaag 480aagctcttac tggcaccagc caacactcag ctcagctaca atgaatgcag cggtcttagc 540actcagaacc atgcagcaaa ccacacccac aggatcagaa caaaccctgc cattgttaag 600accgagaatt catggagcaa taaagcgaag agcatttgtc aacagcaaaa gccacaaaga 660cgtccctgct cagagcttct caagtatctg accacaaacg atgaccctcc tcacaccaaa 720cccacagaaa acaggaacag cagcagagac aaatgtgctt ccaaaaagaa gtcccataca 780caaccgcagt cgcaacatgc tcaagccaaa ccaacaactt tatctcttcc tctgacccca 840gagtcaccaa atgaccccaa gggttcccca tttgagaaca agactattga gcgaacctta 900agtgtggaac tctctggaac tgca 924 5 291 PRT Mus musculus 5 Met Ala Trp AspMet Cys Ser Gln Asp Ser Val Trp Ser Asp Ile Glu 1 5 10 15 Cys Ala AlaLeu Val Gly Glu Asp Gln Pro Leu Cys Pro Asp Leu Pro 20 25 30 Glu Leu AspLeu Ser Glu Leu Asp Val Asn Asp Leu Asp Thr Asp Ser 35 40 45 Phe Leu GlyGly Leu Lys Trp Cys Ser Asp Gln Ser Glu Ile Ile Ser 50 55 60 Asn Gln TyrAsn Asn Glu Pro Ala Asn Ile Phe Glu Lys Ile Asp Glu 65 70 75 80 Glu AsnGlu Ala Asn Leu Leu Ala Val Leu Thr Glu Thr Leu Asp Ser 85 90 95 Leu ProVal Asp Glu Asp Gly Leu Pro Ser Phe Asp Ala Leu Thr Asp 100 105 110 GlyAla Val Thr Thr Asp Asn Glu Ala Ser Pro Ser Ser Met Pro Asp 115 120 125Gly Thr Pro Pro Pro Gln Glu Ala Glu Glu Pro Ser Leu Leu Lys Lys 130 135140 Leu Leu Leu Ala Pro Ala Asn Thr Gln Leu Ser Tyr Asn Glu Cys Ser 145150 155 160 Gly Leu Ser Thr Gln Asn His Ala Ala Asn His Thr His Arg IleArg 165 170 175 Thr Asn Pro Ala Ile Val Lys Thr Glu Asn Ser Trp Ser AsnLys Ala 180 185 190 Lys Ser Ile Cys Gln Gln Gln Lys Pro Gln Arg Arg ProCys Ser Glu 195 200 205 Leu Leu Lys Tyr Leu Thr Thr Asn Asp Asp Pro ProHis Thr Lys Pro 210 215 220 Thr Glu Asn Arg Asn Ser Ser Arg Asp Lys CysAla Ser Lys Lys Lys 225 230 235 240 Ser His Thr Gln Pro Gln Ser Gln HisAla Gln Ala Lys Pro Thr Thr 245 250 255 Leu Ser Leu Pro Leu Thr Pro GluSer Pro Asn Asp Pro Lys Gly Ser 260 265 270 Pro Phe Glu Asn Lys Thr IleGlu Arg Thr Leu Ser Val Glu Leu Ser 275 280 285 Gly Thr Ala 290 6 1893DNA Mus musculus CDS (90)...(1052) 6 gaattcggca cgaggcctgc atgagtgtgtgctgtgtgtc agagtggatt ggagttgaaa 60 aagcttgact ggcgtcattc gggagctgg atggct tgg gac atg tgc agc caa 113 Met Ala Trp Asp Met Cys Ser Gln 1 5 gactct gta tgg agt gac ata gag tgt gct gct ctg gtt ggt gag gac 161 Asp SerVal Trp Ser Asp Ile Glu Cys Ala Ala Leu Val Gly Glu Asp 10 15 20 cag cctctt tgc cca gat ctt cct gaa ctt gac ctt tct gaa ctt gat 209 Gln Pro LeuCys Pro Asp Leu Pro Glu Leu Asp Leu Ser Glu Leu Asp 25 30 35 40 gtg aatgac ttg gat aca gac agc ttt ctg ggt gga ttg aag tgg tgt 257 Val Asn AspLeu Asp Thr Asp Ser Phe Leu Gly Gly Leu Lys Trp Cys 45 50 55 agc gac caatcg gaa atc ata tcc aac cag tac aac aat gag cct gcg 305 Ser Asp Gln SerGlu Ile Ile Ser Asn Gln Tyr Asn Asn Glu Pro Ala 60 65 70 aac ata ttt gagaag ata gat gaa gag aat gag gcg aac ttg cta gcg 353 Asn Ile Phe Glu LysIle Asp Glu Glu Asn Glu Ala Asn Leu Leu Ala 75 80 85 gtc ctc aca gag acactg gac agt ctc ccc gtg gat gaa gac gga ttg 401 Val Leu Thr Glu Thr LeuAsp Ser Leu Pro Val Asp Glu Asp Gly Leu 90 95 100 ccc tca ttt gat gcactg aca gat gga gcc gtg acc act gac aac gag 449 Pro Ser Phe Asp Ala LeuThr Asp Gly Ala Val Thr Thr Asp Asn Glu 105 110 115 120 gcc agt cct tcctcc atg cct gac ggc acc cct ccc cct cag gag gca 497 Ala Ser Pro Ser SerMet Pro Asp Gly Thr Pro Pro Pro Gln Glu Ala 125 130 135 gaa gag ccg tctcta ctt aag aag ctc tta ctg gca cca gcc aac act 545 Glu Glu Pro Ser LeuLeu Lys Lys Leu Leu Leu Ala Pro Ala Asn Thr 140 145 150 cag ctc agc tacaat gaa tgc agc ggt ctt agc act cag aac cat gca 593 Gln Leu Ser Tyr AsnGlu Cys Ser Gly Leu Ser Thr Gln Asn His Ala 155 160 165 gca aac cac acccac agg atc aga aca aac cct gcc att gtt aag acc 641 Ala Asn His Thr HisArg Ile Arg Thr Asn Pro Ala Ile Val Lys Thr 170 175 180 gag aat tca tggagc aat aaa gcg aag agc att tgt caa cag caa aag 689 Glu Asn Ser Trp SerAsn Lys Ala Lys Ser Ile Cys Gln Gln Gln Lys 185 190 195 200 cca caa agacgt ccc tgc tca gag ctt ctc aag tat ctg acc aca aac 737 Pro Gln Arg ArgPro Cys Ser Glu Leu Leu Lys Tyr Leu Thr Thr Asn 205 210 215 gat gac cctcct cac acc aaa ccc aca gaa aac agg aac agc agc aga 785 Asp Asp Pro ProHis Thr Lys Pro Thr Glu Asn Arg Asn Ser Ser Arg 220 225 230 gac aaa tgtgct tcc aaa aag aag tcc cat aca caa ccg cag tcg caa 833 Asp Lys Cys AlaSer Lys Lys Lys Ser His Thr Gln Pro Gln Ser Gln 235 240 245 cat gct caagcc aaa cca aca act tta tct ctt cct ctg acc cca gag 881 His Ala Gln AlaLys Pro Thr Thr Leu Ser Leu Pro Leu Thr Pro Glu 250 255 260 tca cca aatgac ccc aag ggt tcc cca ttt gag aac aag act att gag 929 Ser Pro Asn AspPro Lys Gly Ser Pro Phe Glu Asn Lys Thr Ile Glu 265 270 275 280 cga acctta agt gtg gaa ctc tct gga act gca gct cca cta gtg cca 977 Arg Thr LeuSer Val Glu Leu Ser Gly Thr Ala Ala Pro Leu Val Pro 285 290 295 agg gagcat cca tgc atc att aca tcc agg tcg ata ttg aat gtc ttc 1025 Arg Glu HisPro Cys Ile Ile Thr Ser Arg Ser Ile Leu Asn Val Phe 300 305 310 atg caaaga tgt ctt tct aat tta taa atatgaacac atcacacaac 1072 Met Gln Arg CysLeu Ser Asn Leu * 315 320 ttgtgttcat tctattaaag gtgtaaaaac taatttgatttcaaaatagc tgttgttagt 1132 aaagcaagat gagagaaagg agaatgttct tgtggcagaaggcatttaaa tctattgcat 1192 atggagattt tttttcagac actaccaaca ggattttatgtctgaaatgg aaatggaaag 1252 gcaatgtcag cctaacaagg tgatggcttg aaacacaagacatgaaggaa ctttgttagg 1312 gaccaaaata actggtcccc aattttatgt atatacatacatgttttggc tatcactata 1372 aacatggtga aagcaatgga gctgttttat aactgataaaaagatgaata gaacaaaata 1432 accagctgtc tttttactct cggaccactg ggttctgcccatatttcctt ccattcacat 1492 atctttggtt accttgtttg aaatggggta gacatgcggttaatttggtt tgttattata 1552 ttatttgttt gaggatttca taaataagtg caatatatttgcatcatttc caccccaaca 1612 cctcccaaaa ccacccatct caaattcatt tactctttttctataattgt ttttgtcata 1672 tattacacac acacaaaggc gcatacacac acacgcacacacaggcacac acacacacac 1732 acacacacac acacacacac acacacactg agagttgccctaatttaggg ttgaccactt 1792 agggttcagg tctcatccct gaaaaatgaa gaagaagaagaagaagaaga agaagaagaa 1852 gaagaagaag aagaagaaga agaagaaaaa aaaaaaaaaa a1893 7 320 PRT Mus musculus 7 Met Ala Trp Asp Met Cys Ser Gln Asp SerVal Trp Ser Asp Ile Glu 1 5 10 15 Cys Ala Ala Leu Val Gly Glu Asp GlnPro Leu Cys Pro Asp Leu Pro 20 25 30 Glu Leu Asp Leu Ser Glu Leu Asp ValAsn Asp Leu Asp Thr Asp Ser 35 40 45 Phe Leu Gly Gly Leu Lys Trp Cys SerAsp Gln Ser Glu Ile Ile Ser 50 55 60 Asn Gln Tyr Asn Asn Glu Pro Ala AsnIle Phe Glu Lys Ile Asp Glu 65 70 75 80 Glu Asn Glu Ala Asn Leu Leu AlaVal Leu Thr Glu Thr Leu Asp Ser 85 90 95 Leu Pro Val Asp Glu Asp Gly LeuPro Ser Phe Asp Ala Leu Thr Asp 100 105 110 Gly Ala Val Thr Thr Asp AsnGlu Ala Ser Pro Ser Ser Met Pro Asp 115 120 125 Gly Thr Pro Pro Pro GlnGlu Ala Glu Glu Pro Ser Leu Leu Lys Lys 130 135 140 Leu Leu Leu Ala ProAla Asn Thr Gln Leu Ser Tyr Asn Glu Cys Ser 145 150 155 160 Gly Leu SerThr Gln Asn His Ala Ala Asn His Thr His Arg Ile Arg 165 170 175 Thr AsnPro Ala Ile Val Lys Thr Glu Asn Ser Trp Ser Asn Lys Ala 180 185 190 LysSer Ile Cys Gln Gln Gln Lys Pro Gln Arg Arg Pro Cys Ser Glu 195 200 205Leu Leu Lys Tyr Leu Thr Thr Asn Asp Asp Pro Pro His Thr Lys Pro 210 215220 Thr Glu Asn Arg Asn Ser Ser Arg Asp Lys Cys Ala Ser Lys Lys Lys 225230 235 240 Ser His Thr Gln Pro Gln Ser Gln His Ala Gln Ala Lys Pro ThrThr 245 250 255 Leu Ser Leu Pro Leu Thr Pro Glu Ser Pro Asn Asp Pro LysGly Ser 260 265 270 Pro Phe Glu Asn Lys Thr Ile Glu Arg Thr Leu Ser ValGlu Leu Ser 275 280 285 Gly Thr Ala Ala Pro Leu Val Pro Arg Glu His ProCys Ile Ile Thr 290 295 300 Ser Arg Ser Ile Leu Asn Val Phe Met Gln ArgCys Leu Ser Asn Leu 305 310 315 320 8 960 DNA Mus musculus CDS(1)...(960) 8 atg gct tgg gac atg tgc agc caa gac tct gta tgg agt gacata gag 48 Met Ala Trp Asp Met Cys Ser Gln Asp Ser Val Trp Ser Asp IleGlu 1 5 10 15 tgt gct gct ctg gtt ggt gag gac cag cct ctt tgc cca gatctt cct 96 Cys Ala Ala Leu Val Gly Glu Asp Gln Pro Leu Cys Pro Asp LeuPro 20 25 30 gaa ctt gac ctt tct gaa ctt gat gtg aat gac ttg gat aca gacagc 144 Glu Leu Asp Leu Ser Glu Leu Asp Val Asn Asp Leu Asp Thr Asp Ser35 40 45 ttt ctg ggt gga ttg aag tgg tgt agc gac caa tcg gaa atc ata tcc192 Phe Leu Gly Gly Leu Lys Trp Cys Ser Asp Gln Ser Glu Ile Ile Ser 5055 60 aac cag tac aac aat gag cct gcg aac ata ttt gag aag ata gat gaa240 Asn Gln Tyr Asn Asn Glu Pro Ala Asn Ile Phe Glu Lys Ile Asp Glu 6570 75 80 gag aat gag gcg aac ttg cta gcg gtc ctc aca gag aca ctg gac agt288 Glu Asn Glu Ala Asn Leu Leu Ala Val Leu Thr Glu Thr Leu Asp Ser 8590 95 ctc ccc gtg gat gaa gac gga ttg ccc tca ttt gat gca ctg aca gat336 Leu Pro Val Asp Glu Asp Gly Leu Pro Ser Phe Asp Ala Leu Thr Asp 100105 110 gga gcc gtg acc act gac aac gag gcc agt cct tcc tcc atg cct gac384 Gly Ala Val Thr Thr Asp Asn Glu Ala Ser Pro Ser Ser Met Pro Asp 115120 125 ggc acc cct ccc cct cag gag gca gaa gag ccg tct cta ctt aag aag432 Gly Thr Pro Pro Pro Gln Glu Ala Glu Glu Pro Ser Leu Leu Lys Lys 130135 140 ctc tta ctg gca cca gcc aac act cag ctc agc tac aat gaa tgc agc480 Leu Leu Leu Ala Pro Ala Asn Thr Gln Leu Ser Tyr Asn Glu Cys Ser 145150 155 160 ggt ctt agc act cag aac cat gca gca aac cac acc cac agg atcaga 528 Gly Leu Ser Thr Gln Asn His Ala Ala Asn His Thr His Arg Ile Arg165 170 175 aca aac cct gcc att gtt aag acc gag aat tca tgg agc aat aaagcg 576 Thr Asn Pro Ala Ile Val Lys Thr Glu Asn Ser Trp Ser Asn Lys Ala180 185 190 aag agc att tgt caa cag caa aag cca caa aga cgt ccc tgc tcagag 624 Lys Ser Ile Cys Gln Gln Gln Lys Pro Gln Arg Arg Pro Cys Ser Glu195 200 205 ctt ctc aag tat ctg acc aca aac gat gac cct cct cac acc aaaccc 672 Leu Leu Lys Tyr Leu Thr Thr Asn Asp Asp Pro Pro His Thr Lys Pro210 215 220 aca gaa aac agg aac agc agc aga gac aaa tgt gct tcc aaa aagaag 720 Thr Glu Asn Arg Asn Ser Ser Arg Asp Lys Cys Ala Ser Lys Lys Lys225 230 235 240 tcc cat aca caa ccg cag tcg caa cat gct caa gcc aaa ccaaca act 768 Ser His Thr Gln Pro Gln Ser Gln His Ala Gln Ala Lys Pro ThrThr 245 250 255 tta tct ctt cct ctg acc cca gag tca cca aat gac ccc aagggt tcc 816 Leu Ser Leu Pro Leu Thr Pro Glu Ser Pro Asn Asp Pro Lys GlySer 260 265 270 cca ttt gag aac aag act att gag cga acc tta agt gtg gaactc tct 864 Pro Phe Glu Asn Lys Thr Ile Glu Arg Thr Leu Ser Val Glu LeuSer 275 280 285 gga act gca gct cca cta gtg cca agg gag cat cca tgc atcatt aca 912 Gly Thr Ala Ala Pro Leu Val Pro Arg Glu His Pro Cys Ile IleThr 290 295 300 tcc agg tcg ata ttg aat gtc ttc atg caa aga tgt ctt tctaat tta 960 Ser Arg Ser Ile Leu Asn Val Phe Met Gln Arg Cys Leu Ser AsnLeu 305 310 315 320 9 931 DNA Mus musculus 9 gctccactag tgccaagggagcatccatgc atcattacat ccaggtcgat attgaatgtc 60 ttcatgcaaa gatgtctttctaatttataa atatgaacac atcacacaac ttgtgttcat 120 tctattaaag gtgtaaaaactaatttgatt tcaaaatagc tgttgttagt aaagcaagat 180 gagagaaagg agaatgttcttgtggcagaa ggcatttaaa tctattgcat atggagattt 240 tttttcagac actaccaacaggattttatg tctgaaatgg aaatggaaag gcaatgtcag 300 cctaacaagg tgatggcttgaaacacaaga catgaaggaa ctttgttagg gaccaaaata 360 actggtcccc aattttatgtatatacatac atgttttggc tatcactata aacatggtga 420 aagcaatgga gctgttttataactgataaa aagatgaata gaacaaaata accagctgtc 480 tttttactct cggaccactgggttctgccc atatttcctt ccattcacat atctttggtt 540 accttgtttg aaatggggtagacatgcggt taatttggtt tgttattata ttatttgttt 600 gaggatttca taaataagtgcaatatattt gcatcatttc caccccaaca cctcccaaaa 660 ccacccatct caaattcatttactcttttt ctataattgt ttttgtcata tattacacac 720 acacaaaggc gcatacacacacacgcacac acaggcacac acacacacac acacacacac 780 acacacacac acacacactgagagttgccc taatttaggg ttgaccactt agggttcagg 840 tctcatccct gaaaaatgaagaagaagaag aagaagaaga agaagaagaa gaagaagaag 900 aagaagaaga agaagaaaaaaaaaaaaaaa a 931 10 29 PRT Mus musculus 10 Ala Pro Leu Val Pro Arg GluHis Pro Cys Ile Ile Thr Ser Arg Ser 1 5 10 15 Ile Leu Asn Val Phe MetGln Arg Cys Leu Ser Asn Leu 20 25 11 87 DNA Mus musculus CDS (1)...(87)11 gct cca cta gtg cca agg gag cat cca tgc atc att aca tcc agg tcg 48Ala Pro Leu Val Pro Arg Glu His Pro Cys Ile Ile Thr Ser Arg Ser 1 5 1015 ata ttg aat gtc ttc atg caa aga tgt ctt tct aat tta 87 Ile Leu AsnVal Phe Met Gln Arg Cys Leu Ser Asn Leu 20 25 12 1744 DNA Mus musculusCDS (67)...(969) misc_feature 1543 n = A,T,C or G 12 gaattcggcacgaggtcaga gtggattgga gttgaaaaag cttgactggc gtcattcggg 60 agctgg atg gcttgg gac atg tgc agc caa gac tct gta tgg agt gac 108 Met Ala Trp Asp MetCys Ser Gln Asp Ser Val Trp Ser Asp 1 5 10 ata gag tgt gct gct ctg gttggt gag gac cag cct ctt tgc cca gat 156 Ile Glu Cys Ala Ala Leu Val GlyGlu Asp Gln Pro Leu Cys Pro Asp 15 20 25 30 ctt cct gaa ctt gac ctt tctgaa ctt gat gtg aat gac ttg gat aca 204 Leu Pro Glu Leu Asp Leu Ser GluLeu Asp Val Asn Asp Leu Asp Thr 35 40 45 gac agc ttt ctg ggt gga ttg aagtgg tgt agc gac caa tcg gaa atc 252 Asp Ser Phe Leu Gly Gly Leu Lys TrpCys Ser Asp Gln Ser Glu Ile 50 55 60 ata tcc aac cag tac aac aat gag cctgcg aac ata ttt gag aag ata 300 Ile Ser Asn Gln Tyr Asn Asn Glu Pro AlaAsn Ile Phe Glu Lys Ile 65 70 75 gat gaa gag aat gag gca aac ttg cta gcggtc ctc aca gag aca ctg 348 Asp Glu Glu Asn Glu Ala Asn Leu Leu Ala ValLeu Thr Glu Thr Leu 80 85 90 gac agt ctc ccc gtg gat gaa gac gga ttg ccctca ttt gat gca ctg 396 Asp Ser Leu Pro Val Asp Glu Asp Gly Leu Pro SerPhe Asp Ala Leu 95 100 105 110 aca gat gga gcc gtg acc act gac aac gaggcc agt cct tcc tcc atg 444 Thr Asp Gly Ala Val Thr Thr Asp Asn Glu AlaSer Pro Ser Ser Met 115 120 125 cct gac ggc acc cct ccc cct cag gag gcagaa gag ccg tct cta ctt 492 Pro Asp Gly Thr Pro Pro Pro Gln Glu Ala GluGlu Pro Ser Leu Leu 130 135 140 aag aag ctc tta ctg gca cca gcc aac actcag ctc agc tac aat gaa 540 Lys Lys Leu Leu Leu Ala Pro Ala Asn Thr GlnLeu Ser Tyr Asn Glu 145 150 155 tgc agc ggt ctt agc act cag aac cat gcagca aac cac acc cac agg 588 Cys Ser Gly Leu Ser Thr Gln Asn His Ala AlaAsn His Thr His Arg 160 165 170 atc aga aca aac cct gcc att gtt aag accgag aat tca tgg agc aat 636 Ile Arg Thr Asn Pro Ala Ile Val Lys Thr GluAsn Ser Trp Ser Asn 175 180 185 190 aaa gcg aag agc att tgt caa cag caaaag cca caa aga cgt ccc tgc 684 Lys Ala Lys Ser Ile Cys Gln Gln Gln LysPro Gln Arg Arg Pro Cys 195 200 205 tca gag ctt ctc aag tat ctg acc acaaac gat gac cct cct cac acc 732 Ser Glu Leu Leu Lys Tyr Leu Thr Thr AsnAsp Asp Pro Pro His Thr 210 215 220 aaa ccc aca gaa aac agg aac agc agcaga gac aaa tgt gct tcc aaa 780 Lys Pro Thr Glu Asn Arg Asn Ser Ser ArgAsp Lys Cys Ala Ser Lys 225 230 235 aag aag tcc cat aca caa ccg cag tcgcaa cat gct caa gcc aaa cca 828 Lys Lys Ser His Thr Gln Pro Gln Ser GlnHis Ala Gln Ala Lys Pro 240 245 250 aca act tta tct ctt cct ctg acc ccagag tca cca aat gac ccc aag 876 Thr Thr Leu Ser Leu Pro Leu Thr Pro GluSer Pro Asn Asp Pro Lys 255 260 265 270 ggt tcc cca ttt gag aac aag actatt gag cga acc tta agt gtg gaa 924 Gly Ser Pro Phe Glu Asn Lys Thr IleGlu Arg Thr Leu Ser Val Glu 275 280 285 ctc tct gga act gca ggt gta aaaact aat ttg att tca aaa tag 969 Leu Ser Gly Thr Ala Gly Val Lys Thr AsnLeu Ile Ser Lys * 290 295 300 ctgttgttag ttaagcaaga tgagagaaaggagaatgttc ttgtggcaga aggcatttaa 1029 atctattgca tatggagatt ttttttcagacactaccaac aggattttat gtctgaaatg 1089 gaaatggaaa ggcaatgtca gcctaacaaggtgatggctt gaaacacaag acatgaagga 1149 actttgttag ggaccaaaat aactggtccccaattttatg tatatacata catgttttgg 1209 ctatcactat aaacatggtg aaagcaatggagctgtttta taactgataa aaagatgaat 1269 agaacaaaat aaccagctgt ctttttactctcggaccact gggttctgcc catatttcct 1329 tccattcaca tatctttggt taccttgtttgaaatggggt agacatgcgg ttaatttggt 1389 ttgttattat attatttgtt tgaggatttcataaataagt gcaatatatt tgcatcattt 1449 ccaccccaac acctcccaaa accacccatctcaaattcat ttactctttt tctataattg 1509 tttttgtcat atattacaca cacacaaaggcacntacaca cacacgcaca cacaggcaca 1569 cacacacaca cacacacaca cacacacacacacacacact gagaattgcc ctaatttagg 1629 gttgaccact tagggttcag tttttttccctggaaaatgg ggggggggga aaaaaaaaaa 1689 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaa 1744 13 300 PRT Mus musculus 13 Met Ala TrpAsp Met Cys Ser Gln Asp Ser Val Trp Ser Asp Ile Glu 1 5 10 15 Cys AlaAla Leu Val Gly Glu Asp Gln Pro Leu Cys Pro Asp Leu Pro 20 25 30 Glu LeuAsp Leu Ser Glu Leu Asp Val Asn Asp Leu Asp Thr Asp Ser 35 40 45 Phe LeuGly Gly Leu Lys Trp Cys Ser Asp Gln Ser Glu Ile Ile Ser 50 55 60 Asn GlnTyr Asn Asn Glu Pro Ala Asn Ile Phe Glu Lys Ile Asp Glu 65 70 75 80 GluAsn Glu Ala Asn Leu Leu Ala Val Leu Thr Glu Thr Leu Asp Ser 85 90 95 LeuPro Val Asp Glu Asp Gly Leu Pro Ser Phe Asp Ala Leu Thr Asp 100 105 110Gly Ala Val Thr Thr Asp Asn Glu Ala Ser Pro Ser Ser Met Pro Asp 115 120125 Gly Thr Pro Pro Pro Gln Glu Ala Glu Glu Pro Ser Leu Leu Lys Lys 130135 140 Leu Leu Leu Ala Pro Ala Asn Thr Gln Leu Ser Tyr Asn Glu Cys Ser145 150 155 160 Gly Leu Ser Thr Gln Asn His Ala Ala Asn His Thr His ArgIle Arg 165 170 175 Thr Asn Pro Ala Ile Val Lys Thr Glu Asn Ser Trp SerAsn Lys Ala 180 185 190 Lys Ser Ile Cys Gln Gln Gln Lys Pro Gln Arg ArgPro Cys Ser Glu 195 200 205 Leu Leu Lys Tyr Leu Thr Thr Asn Asp Asp ProPro His Thr Lys Pro 210 215 220 Thr Glu Asn Arg Asn Ser Ser Arg Asp LysCys Ala Ser Lys Lys Lys 225 230 235 240 Ser His Thr Gln Pro Gln Ser GlnHis Ala Gln Ala Lys Pro Thr Thr 245 250 255 Leu Ser Leu Pro Leu Thr ProGlu Ser Pro Asn Asp Pro Lys Gly Ser 260 265 270 Pro Phe Glu Asn Lys ThrIle Glu Arg Thr Leu Ser Val Glu Leu Ser 275 280 285 Gly Thr Ala Gly ValLys Thr Asn Leu Ile Ser Lys 290 295 300 14 900 DNA Mus musculus CDS(1)...(900) 14 atg gct tgg gac atg tgc agc caa gac tct gta tgg agt gacata gag 48 Met Ala Trp Asp Met Cys Ser Gln Asp Ser Val Trp Ser Asp IleGlu 1 5 10 15 tgt gct gct ctg gtt ggt gag gac cag cct ctt tgc cca gatctt cct 96 Cys Ala Ala Leu Val Gly Glu Asp Gln Pro Leu Cys Pro Asp LeuPro 20 25 30 gaa ctt gac ctt tct gaa ctt gat gtg aat gac ttg gat aca gacagc 144 Glu Leu Asp Leu Ser Glu Leu Asp Val Asn Asp Leu Asp Thr Asp Ser35 40 45 ttt ctg ggt gga ttg aag tgg tgt agc gac caa tcg gaa atc ata tcc192 Phe Leu Gly Gly Leu Lys Trp Cys Ser Asp Gln Ser Glu Ile Ile Ser 5055 60 aac cag tac aac aat gag cct gcg aac ata ttt gag aag ata gat gaa240 Asn Gln Tyr Asn Asn Glu Pro Ala Asn Ile Phe Glu Lys Ile Asp Glu 6570 75 80 gag aat gag gca aac ttg cta gcg gtc ctc aca gag aca ctg gac agt288 Glu Asn Glu Ala Asn Leu Leu Ala Val Leu Thr Glu Thr Leu Asp Ser 8590 95 ctc ccc gtg gat gaa gac gga ttg ccc tca ttt gat gca ctg aca gat336 Leu Pro Val Asp Glu Asp Gly Leu Pro Ser Phe Asp Ala Leu Thr Asp 100105 110 gga gcc gtg acc act gac aac gag gcc agt cct tcc tcc atg cct gac384 Gly Ala Val Thr Thr Asp Asn Glu Ala Ser Pro Ser Ser Met Pro Asp 115120 125 ggc acc cct ccc cct cag gag gca gaa gag ccg tct cta ctt aag aag432 Gly Thr Pro Pro Pro Gln Glu Ala Glu Glu Pro Ser Leu Leu Lys Lys 130135 140 ctc tta ctg gca cca gcc aac act cag ctc agc tac aat gaa tgc agc480 Leu Leu Leu Ala Pro Ala Asn Thr Gln Leu Ser Tyr Asn Glu Cys Ser 145150 155 160 ggt ctt agc act cag aac cat gca gca aac cac acc cac agg atcaga 528 Gly Leu Ser Thr Gln Asn His Ala Ala Asn His Thr His Arg Ile Arg165 170 175 aca aac cct gcc att gtt aag acc gag aat tca tgg agc aat aaagcg 576 Thr Asn Pro Ala Ile Val Lys Thr Glu Asn Ser Trp Ser Asn Lys Ala180 185 190 aag agc att tgt caa cag caa aag cca caa aga cgt ccc tgc tcagag 624 Lys Ser Ile Cys Gln Gln Gln Lys Pro Gln Arg Arg Pro Cys Ser Glu195 200 205 ctt ctc aag tat ctg acc aca aac gat gac cct cct cac acc aaaccc 672 Leu Leu Lys Tyr Leu Thr Thr Asn Asp Asp Pro Pro His Thr Lys Pro210 215 220 aca gaa aac agg aac agc agc aga gac aaa tgt gct tcc aaa aagaag 720 Thr Glu Asn Arg Asn Ser Ser Arg Asp Lys Cys Ala Ser Lys Lys Lys225 230 235 240 tcc cat aca caa ccg cag tcg caa cat gct caa gcc aaa ccaaca act 768 Ser His Thr Gln Pro Gln Ser Gln His Ala Gln Ala Lys Pro ThrThr 245 250 255 tta tct ctt cct ctg acc cca gag tca cca aat gac ccc aagggt tcc 816 Leu Ser Leu Pro Leu Thr Pro Glu Ser Pro Asn Asp Pro Lys GlySer 260 265 270 cca ttt gag aac aag act att gag cga acc tta agt gtg gaactc tct 864 Pro Phe Glu Asn Lys Thr Ile Glu Arg Thr Leu Ser Val Glu LeuSer 275 280 285 gga act gca ggt gta aaa act aat ttg att tca aaa 900 GlyThr Ala Gly Val Lys Thr Asn Leu Ile Ser Lys 290 295 300 15 805 DNA Musmusculus misc_feature 604 n = A,T,C or G 15 ggtgtaaaaa ctaatttgatttcaaaatag ctgttgttag ttaagcaaga tgagagaaag 60 gagaatgttc ttgtggcagaaggcatttaa atctattgca tatggagatt ttttttcaga 120 cactaccaac aggattttatgtctgaaatg gaaatggaaa ggcaatgtca gcctaacaag 180 gtgatggctt gaaacacaagacatgaagga actttgttag ggaccaaaat aactggtccc 240 caattttatg tatatacatacatgttttgg ctatcactat aaacatggtg aaagcaatgg 300 agctgtttta taactgataaaaagatgaat agaacaaaat aaccagctgt ctttttactc 360 tcggaccact gggttctgcccatatttcct tccattcaca tatctttggt taccttgttt 420 gaaatggggt agacatgcggttaatttggt ttgttattat attatttgtt tgaggatttc 480 ataaataagt gcaatatatttgcatcattt ccaccccaac acctcccaaa accacccatc 540 tcaaattcat ttactctttttctataattg tttttgtcat atattacaca cacacaaagg 600 cacntacaca cacacgcacacacaggcaca cacacacaca cacacacaca cacacacaca 660 cacacacact gagaattgccctaatttagg gttgaccact tagggttcag tttttttccc 720 tggaaaatgg gggggggggaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 780 aaaaaaaaaa aaaaaaaaaaaaaaa 805 16 9 PRT Mus musculus 16 Gly Val Lys Thr Asn Leu Ile Ser Lys 15 17 27 DNA Mus musculus CDS (1)...(27) 17 ggt gta aaa act aat ttg atttca aaa 27 Gly Val Lys Thr Asn Leu Ile Ser Lys 1 5

What is claimed:
 1. An isolated nucleic acid molecule selected from thegroup consisting of: a) a nucleic acid molecule comprising thenucleotide sequence set forth in SEQ ID NO:6 or SEQ ID NO:12, or acomplement thereof; b) a nucleic acid molecule comprising the nucleotidesequence set forth in SEQ ID NO:8 or SEQ ID NO:14, or a complementthereof; c) a nucleic acid molecule comprising the nucleotide sequenceset forth in SEQ ID NO:9 or SEQ ID NO:15, or a complement thereof; andd) a nucleic acid molecule comprising the nucleotide sequence set forthin SEQ ID NO:11 or SEQ ID NO:17, or a complement thereof.
 2. An isolatednucleic acid molecule which encodes a polypeptide comprising the aminoacid sequence set forth in SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, orSEQ ID NO:16, or a complement thereof
 3. An isolated nucleic acidmolecule comprising the nucleotide sequence contained in the plasmiddeposited with ATCC® as Accession Number ______ or ______.
 4. Anisolated nucleic acid molecule selected from the group consisting of: a)a nucleic acid molecule comprising a nucleotide sequence which is atleast 60% identical to the nucleotide sequence of SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:15, or SEQ ID NO:17, or a complement thereof; b) a nucleic acidmolecule comprising a fragment of at least 30 nucleotides of a nucleicacid comprising the nucleotide sequence of SEQ ID NO:6, SEQ ID NO:8, SEQID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, or SEQID NO:17, or a complement thereof; c) a nucleic acid molecule whichencodes a polypeptide comprising an amino acid sequence at least about60% identical to the amino acid sequence of SEQ ID NO:7, SEQ ID NO:10,SEQ ID NO:13, or SEQ ID NO:16, or a complement thereof; and d) a nucleicacid molecule which encodes a fragment of a polypeptide comprising theamino acid sequence of SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, or SEQID NO:16, wherein the fragment comprises at least 10 contiguous aminoacid residues of the amino acid sequence of SEQ ID NO:7, SEQ ID NO:10,SEQ ID NO:13, or SEQ ID NO:16, or a complement thereof.
 5. The isolatednucleic acid molecule of claim 4, wherein the nucleic acid moleculeencodes a polypeptide which is sufficiently identical to the polypeptideof SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, or SEQ ID NO:16, such thatthe polypeptide maintains the ability to modulate one or more of thefollowing biological activities: interaction with a nuclear receptor,UCP-2 expression, Acyl Co-A Oxidase expression, thermogenesis in adiposecells, differentiation of adipose cells, and insulin sensitivity ofadipose cells.
 6. The isolated nucleic acid molecule of claim 5, whereinthe polypeptide comprises one or more of the following domains ormotifs: a) a cAMP phosphorylation site; b) a tyrosine phosphorylationsite; and c) an LXXLL motif.
 7. An isolated nucleic acid molecule whichhybridizes to the nucleic acid molecule of any one of claims 1, 2, 3, 4,5, or 6 under stringent conditions.
 8. An isolated nucleic acid moleculecomprising the nucleic acid molecule of any one of claims 1, 2, 3, 4, 5,or 6, and a nucleotide sequence encoding a heterologous polypeptide. 9.A vector comprising the nucleic acid molecule of any one of claims 1, 2,3, 4, 5, or
 6. 10. The vector of claim 9, which is an expression vector.11. A host cell transfected with the expression vector of claim
 10. 12.A method of producing a polypeptide comprising culturing the host cellof claim 11 in an appropriate culture medium to, thereby, produce thepolypeptide.
 13. An isolated polypeptide selected from the groupconsisting of: a) a fragment of a polypeptide comprising the amino acidsequence of SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, or SEQ ID NO:16,wherein the fragment comprises at least 10 contiguous amino acids of SEQID NO:7, SEQ ID NO:10, SEQ ID NO:13, or SEQ ID NO:16; b) a polypeptidewhich is encoded by a nucleic acid molecule comprising a nucleotidesequence which is at least 60% identical to a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, or SEQ IDNO:17; and c) a polypeptide comprising an amino acid sequence which isat least 60% identical to the amino acid sequence of SEQ ID NO:7, SEQ IDNO:10, SEQ ID NO:13, or SEQ ID NO:16.
 14. The isolated polypeptide ofclaim 13, wherein the polypeptide maintains the ability to modulate oneor more of the following biological activities: interaction with anuclear receptor, UCP-2 expression, Acyl Co-A Oxidase expression,thermogenesis in adipose cells, differentiation of adipose cells, andinsulin sensitivity of adipose cells.
 15. The isolated polypeptide ofclaim 14, wherein the polypeptide comprises one or more of the followingdomains or motifs: a) a cAMP phosphorylation site; b) a tyrosinephosphorylation site; and c) an LXXLL motif.
 16. The isolatedpolypeptide of claim 13 comprising the amino acid sequence of SEQ IDNO:7, SEQ ID NO:10, SEQ ID NO:13, or SEQ ID NO:16.
 17. The polypeptideof claim 13, further comprising heterologous amino acid sequences. 18.An antibody which selectively binds to a polypeptide of claim
 13. 19. Amethod for detecting the presence of a polypeptide of claim 13 in asample comprising: a) contacting the sample with a compound whichselectively binds to the polypeptide; and b) determining whether thecompound binds to the polypeptide in the sample to thereby detect thepresence of a polypeptide of claim 13 in the sample.
 20. The method ofclaim 19, wherein the compound which binds to the polypeptide is anantibody.
 21. A kit comprising a compound which selectively binds to apolypeptide of claim 13 and instructions for use.
 22. A method fordetecting the presence of a nucleic acid molecule of any one of claims1, 2, 3, 4, 5, or 6 in a sample comprising: a) contacting the samplewith a nucleic acid probe or primer which selectively hybridizes to thenucleic acid molecule; and b) determining whether the nucleic acid probeor primer binds to a nucleic acid molecule in the sample to therebydetect the presence of a nucleic acid molecule of any one of claims 1,2, 3, 4, 5, or 6 in the sample.
 23. The method of claim 22, wherein thesample comprises mRNA molecules and is contacted with a nucleic acidprobe.
 24. A kit comprising the nucleic acid molecule of any one ofclaims 1, 2, 3, 4, 5, or 6 and instructions for use.
 25. A method foridentifying a compound which binds to a polypeptide of claim 13comprising: a) contacting the polypeptide, or a cell expressing thepolypeptide with a test compound, and b) determining whether thepolypeptide binds to the test compound.
 26. The method of claim 25,wherein the binding of the test compound to the polypeptide is detectedby a method selected from the group consisting of: a) detection ofbinding by direct detection of test compound/polypeptide binding; b)detection of binding using a competition binding assay; c) detection ofbinding using an assay for PGC-1b activity; and c) detection of bindingusing an assay for PGC-1c activity.
 27. The method of claim 26, whereinPGC-1b activity is selected from the group consisting of: a)interaction, with a nuclear receptor, b) modulation of UCP-2 expression;c) modulation of Acyl Co-A Oxidase expression; d) modulation ofthermogenesis in adipose cells; e) modulation of differentiation ofadipose cells; and f) modulation of insulin sensitivity of adiposecells.
 28. The method of claim 26, wherein PGC-1c activity is selectedfrom the group consisting of: a) interaction with a nuclear receptor; b)modulation of UCP-2 expression; c) modulation of Acyl Co-A Oxidaseexpression; d) modulation of thermogenesis in adipose cells; e)modulation of differentiation of adipose cells; and f) modulation ofinsulin sensitivity of adipose cells.
 29. A method for modulating theactivity of a polypeptide of claim 13 comprising contacting thepolypeptide or a cell expressing the polypeptide with a compound whichbinds to the polypeptide in a sufficient concentration to modulate theactivity of the polypeptide.
 30. The method of any of claims 25, 26, or27, herein the cell is selected from the group consisting of a myoblast,a differentiated myoblast, a muscle cell, a preadipose cell, and anadipose cell.
 31. A method for identifying a compound which modulatesthe activity of a polypeptide of claim 13 comprising: a) contacting apolypeptide of claim 13 with a test compound; and b) determining theeffect of the test compound on the activity of the polypeptide tothereby identify a compound which modulates the activity of thepolypeptide.
 32. The method of claim 31, wherein the activity of thepolypeptide is selected from the group consisting of: a) interactionwith a nuclear receptor; b) modulation of UCP-2 expression; c)modulation of Acyl Co-A Oxidase expression; d) modulation ofthermogenesis in adipose cells; e) modulation of differentiation ofadipose cells; and f) modulation of insulin sensitivity of adiposecells.